Chemically induced intracellular hyperthermia

ABSTRACT

An invention relating to therapeutic pharmacological agents and methods to chemically induce intracellular hyperthermia and/or free radicals for the diagnosis and treatment of infections, malignancy and other medical conditions. The invention relates to a process and composition for the diagnosis or killing of cancer cells and inactivation of susceptible bacterial, parasitic, fungal, and viral pathogens by chemically generating heat, and/or free radicals and/or hyperthermia—inducible immunogenic determinants by using mitochondrial uncoupling agents, especially 2,4 dinitrophenol and, their conjugates, either alone or in combination with other drugs, hormones, cytokines and radiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. §120 ofcopending U.S. patent application Ser. No. 12/613,189, filed Nov. 5,2009, which is a continuation of U.S. patent application Ser. No.09/744,622, filed Jan. 26, 2001, now U.S. Pat. No. 7,635,722, issuedDec. 22, 2009, which is a national stage application of internationalpatent application PCT/US1999/16940 filed Jul. 27, 1999, which claimsunder 35 U.S.C. §119(e) the benefit of U.S. provisional patentapplication 60/094,286, filed Jul. 27, 1998, all of which applicationsare hereby incorporated by reference in their entireties.

FIELD OF INVENTION

This invention relates to therapeutic pharmacological agents and methodsto chemically induce intracellular hyperthermia and/or free radicals forthe diagnosis and treatment of infections, malignancy and other medicalconditions. This invention further relates to a process and compositionfor the diagnosis or killing of cancer cells and inactivation ofsusceptible bacterial, parasitic, fungal, and viral pathogens bychemically generating heat, free radicals and hyperthermia-inducibleimmunogenic determinants. Such pathogens, infected or transformed cellsare inactivated or killed without irreparable injury to non-transformed,uninfected, normal cells. More specifically, this invention relates tothe diagnosis and treatment of cancer; treatment of AIDS; and, otherdiseases and conditions using mitochondrial uncoupling agents,especially 2,4 dinitrophenol and, their conjugates, either alone or incombination with other drugs, hormones, cytokines and radiation.

GENERAL BACKGROUND

Local heat, systemic hyperthermia and fever therapy have beenempirically used as effective treatments for malignant, infectious andother diseases since antiquity. Therapeutic hyperthermia was firstdocumented in the Edwin Smith surgical papyrus in the 17th century B.C.Coley's toxin extracts of Streptococcus erysipelatis (group Astreptococcus) and Bacillus prodigiosus (Serratia marcescens) were usedto induce fever for the treatment of patients with advanced cancer. TheNobel Prize was awarded for using fever therapy in the treatment ofneurosyphilis with the injection of malarial blood. As late as 1955, theMayo Clinic advocated using malariotherapy or heat therapy for cases oftertiary syphilis “resistant to penicillin” Long term remissions inpatients with inoperable carcinomas that were treated with hot baths andlocal heat applications have also been reported. Published observationson the disappearance of malignancies such as a soft tissue sarcoma in apatient experiencing high fever due to erysipelas and tumor lysis ofBurkitt's lymphomas following malignant hyperthermia during surgicalanesthesia are known. A comprehensive historical review on anecdotalobservations and intuitive rational for the empirical use of therapeutichyperthermia has been published by Myer, J. L.

The temperature of a body can be intentionally increased either bypyrogens to produce fever (fever therapy) or, by the induction ofhyperthermia (therapeutic hyperthermia). Fever raises body temperatureby elevating the thermoregulatory “set point” located in the preopticregion of the anterior hypothalamus. In so doing, the bodyphysiologically works to maintain the higher temperature setting. Theelevated core body temperature increased by fever may or may not beraised above the higher set point value. In contrast, inducedhyperthermia always raises the body temperature above the hypothalamicthermoregulatory set point and the physiologically intact body attemptsto lower it's core temperature back to the set point baseline.

Renewed clinical interest in hyperthermia has occurred over the past 35years due to continued failure of standard therapies to treat variousforms of cancer and emerging infections. Except for a few exceedinglyrare forms of cancer like childhood leukemias and testicular cancer orimmune responsive infections, chemotherapy, radiation or drug therapyoften do very little except briefly extend survival. One of the majorobstacles to “cure” disseminated cancer and infections has been theinnate or acquired resistance of tumor cells and emerging microbes toantibiotics, drugs and treatments given in tolerable doses. Escalationof treatments, or use of multiple drugs to overcome resistance isinvariably prevented by concomitant toxicities or development ofmulti-drug resistance. Further, in contrast to drugs, which represent asingle molecular species that biochemically interact with specificenzymes or receptors of viruses, prokaryotes and eukaryotes, the actionof hyperthermia is biophysical and global. Hyperthermia has no specificheat receptors. Therefore, the possibility of a point mutation causing afunctional change in a receptor and conferring resistance tohyperthermia is unlikely, and would be equivalent to the development ofresistance to the in vitro process of Pasteurization. Among pathogenicbacteria, it has been reported that only one variant in 1×10⁶ cells ofan original population is resistant to hyperthermia.

Hyperthermia has been used alone or in conjunction with radiation andchemotherapy in the treatment of a variety of malignancies. Overgaard etal., reported that a combination of heat and radiation results incomplete control of twice as many melanoma lesions compared to radiationalone. Maeda, M., Watanabe, N. et al., published in GastroenterologiaJaponica, that hyperthermia with tumor necrosis factor resulted insuccessful treatment of hepatocellular carcinoma. Prospective randomizedstudies of hyperthermia combined with chemoradiotherapy for esophagealcarcinoma demonstrated the cumulative three year survival rates to bemore than doubled with the addition of hyperthermia tochemoradiotherapy. Combination chemotherapy with hyperthermia inmetastatic breast cancer refractory to common therapies, i.e., failedprior hormonal therapy and chemotherapy, resulted in 39% completeremissions and 23% partial remissions: relief of bone pain was strikingFujimoto, S., Takahashi, M. et al., demonstrated that the 5 yearsurvival rate of patients with peritoneal carcinomatosis from gastriccarcinoma treated with intraperitoneal hyperthermic chemoperfusion was41.6%, whereas the 50% survival duration of the group that did notreceive intraperitoneal hyperthermia was 110 days. Preoperativehyperthermia with chemotherapy and radiation is also known to improvelong-term results in patients with carcinoma of the rectum, especiallythose with advanced disease. It is clinically known that regional, i.e.,limb, hyperthermic perfusions with chemotherapy is useful for thetreatment of melanoma. Combination therapy with hyperthermia andradiation has been successful in the treatment of non-Hodgkinslymphomas. More recently, a survival benefit of hyperthermia was shownin a prospective randomized trial for patients with glioblastomamultiforme undergoing radiotherapy. However, rigorous clinicalprospective randomized trials with hyperthermia alone or, in combinationwith agents outside its use with radiation therapy have not beenperformed.

The scientific rationale for therapeutic hyperthermia in cancer therapyrests on known data from pre-clinical, in vitro and animal studies.Tumor cells in tissue culture have been demonstrated to be directly moresensitive to heat as compared to their non-malignant counterparts. Cellsundergoing mitosis, synthesizing DNA in the ‘S-phase’, are especiallymore sensitive to hyperthermia. Human leukemic progenitor cells havebeen shown to be selectively killed by hyperthermia and, such in vitrouse has been shown to purge bone marrow of residual tumor cells beforeautologous bone marrow transplantation. Microcalorimetric measurementsconfirm that tumorous tissues produce more heat and are “hotter” thantheir non-tumorous counterparts. As a consequence, they are less able totolerate additional heat loads.

Tumor cells are also killed by heat indirectly. Tumor angiogenesis isinhibited by heat. Hyperthermia causes tumors to have increased heatretention with increased cytoxicity due to tumor neovasculature lackingsmooth muscle and vessel wall precursors needed for cooling byvasodilation. Increased hypoxia, acidity, Fos gene death signaling,decreased nutrient supply and enhanced immunologic cytotoxicity havealso been reported to be caused by hyperthermia and contribute toenhanced tumor cell death. Further, the combination of hyperthermia withchemotherapy and/or radiation has been shown to be supraadditive orsynergistic on killing of tumors. Human gastric carcinoma cells havebeen shown to be selectively killed by a combination of cisplatin, tumornecrosis factor and hyperthermia: a 40% increase in cisplatin DNA damagewas noted in the presence of the three agent combination over cisplatinalone or either dual combination. Numerous animal studies, including theinitial publication by Crile, show that neoplasms transplanted into miceregress when treated with hyperthermia without irreparable damage toadjacent tissues.

Body temperature is a critical factor in determining hostsusceptibility, location of lesions, and the natural history of manyinfectious diseases. Temperature has direct effects on the growth of allmicroorganisms, including those that are pathogenic. Almost all of thebacteria that cause disease in humans grow optimally within the range of33-41° C. and, their temperature growth characteristics are not easilyaltered in vitro. By example, the lesions of Hansen's disease (leprosy)caused by Mycobacterium leprae, characteristically grow and destroy themost acral, coolest parts of the body such as fingers, toes, externalear, the air-stream cooled nasal alae and larynx. Leprosy organismsproliferate and follow the coolest temperature gradients in the body,25-33° C. In animals, the leprae organisms can only be grown in thearmadillo or foot pads of mice were the in situ lesion temperatures are27-30° C. Spontaneous improvement in leprosy lesions have been reportedin patients following febrile illness. Fever therapy, hot baths andlocal heat therapy were formerly utilized in treating this disease.Hyperthermia is also known to destroy Treponema pallidum, the causativeagent of syphilis, by heating five hours at 39° C., three hours at 40°C., two hours at 41° C. or one hour at 41.5° C. The spirochetesresponsible for yaws, bejel, pinta and Lyme disease show similartemperature sensitivity.

Other bacteria that predominately cause lesions at cool sites and aresusceptible to heat inactivation include, Neisseria gonorrhea,Hemophillus ducrei (chancroid), Mycobacterium ulcerans, Mycobacteriummarinum (“swimming pool” granuloma), Diptheria, etc. Further,hyperthermia has been reported to be synergistic with antibiotic andchemotherapy in the treatment of various bacterial diseases. Elevatedbody temperature potentiates the effect of penicillin on stapholococciand syphilis. Hyperthermia makes sulfadiazene bactericidal forstreptococci. Moreover, recent controlled studies show that whenantipyretics are used in animals with severe experimentally inducedinfections, there is increased mortality. Nonetheless, systemichyperthermia has generally been abandoned as a treatment for bacterialinfections with the advent of antibiotics.

Hyperthermia has remained an effective treatment for many fungalinfections. Superficial dermatophytosis flourish in cooler regions ofthe body and heat treatment is oftentimes the only viable therapy fortheir chronic granulomatus lesions. By example, Sporothrix schenkii, thecausative agent of sporotrichosis, has a temperature growth optimum wellbelow 37° C. and is successfully eliminated by local hyperthermia.Similarly, patients with pseudallescheriosis unresponsive to antifungalantibiotics are healed with hyperthermic treatments. In Japan, pocketwarmers, hot water and infrared heating remain current and effectivetreatments for various fungal infections. Systemic hyperthermia,utilizing a Liebel-Flarsheim (Kettering) Hypertherm Fever Cabinet,dramatically treated a case of disseminated sporotrichosis withrecurrent iridocyclitis, repeated post-treatment cultures from thepatient remained negative.

The role of hyperthermia in modulating the clinical course of otherfungal infections, including histoplasmosis, North Americanblastomycosis, chromomycosis, cryptococcosis, paracoccidioidomycosis,Lobos' disease and candidiasis has been described. Fungi, such asNocardia, Actinomyces and Aspergillus also proliferate in cooler regionsof the body causing mandible (lumpy jaw) and foot lesions (Madura foot)respectively. In vitro heat sensitivity data for many of the above andother pathogenic fungi have been reported by Mackinnon et al., Silva andothers.

The effect of temperature and hyperthermia on the pathogenesis ofparasitic disease is also well known. Leishmaniasis, a wide spreadparasitic disease transmitted by the bite of a sandfly, clinicallyinfects 12 million people worldwide. The cutaneous and mucocutaneouslesions, i.e., Oriental sore, Baghdad boil, Delhi boil, Chiclero's ulcerand espundia, are often very destructive and permanently disfiguring.Hyperthermia with moist heat of 39° to 41° C. applied for 20 hours overseveral days has proven to be an effective treatment. In vitro, humanmacrophages infected with Leishmania mexicana are completely destroyedby heating at 39° C. for 3 days. All mucocutaneous Leishmania strains,regardless of subspecies, demonstrate a growth optimum of 35° C. withonly the L. tropica and L. donovani strains surviving temperatures of39° C. Clinical observations have shown that hyperthermic treatment ofone Leishmania lesion often invokes an immune response and results inthe healing of other lesions over a 5-6 week period. The effect ofhyperthermia on other parasites, including Trypanosoma cruzi, malaria,microfilaria, acanthamoeba, trematodes and cestodes has been published.

Increased body temperature is also recognized as a major factor inrecovery from viral infections. Many viruses multiply better attemperatures below 37° C. and their multiplication is inhibited orstopped if the body temperatures exceeds 39° C. In vitro Rhinovirusreplication, for example, falls off by 10⁶ log units with an upwardtemperature shift of 2° C. (37° to 39° C.). Herpes virus replication, aswell as the intracellular and extracellular herpes virus concentration,markedly decrease when the incubation temperature is elevated to 40° C.Varicella virus production in human fibroblastic cell culture is optimalat 37° C. and ceases at 39° C.

Beneficial effects of hyperthermia on the outcome of viral disease inlaboratory animals infected with myxomatosis, encephalomyocarditis,herpes, gastroenteritis, rabies and the common cold in man have beendocumented. Influenza and viruses causing upper respiratory infections,such as the common cold, thrive in a cool body milieu of 30°-35° C.Temperature gradients in this range exist in the fall and winter withinthe oral, nasal, tracheal and laryngeal mucosa and lead to flu andinfluenza epidemics. Live respiratory-virus vaccines for influenza havebeen developed by use of heat-sensitive mutants that cannot reduplicateor cause clinical disease at 36°-37° C. It is known that even as littleas a 0.5° C. difference in the ceiling replication temperature of avirus can have a dramatic effect on virulence and pathogenicity.

Other animal viruses such as Newcastle disease in chickens, rabbitpapilloma, feline leukemia, rabbitpox, hoof-and-mouth disease in cattle,hand, foot, and mouth disease, human plantar warts, and the “grease” ofhorses, due to horsepox involvement of the colder acral extremitiesabove the fetlocks, are known to be very sensitive to inhibition byheat. Heat treatment of cells infected with human immunodeficiency virus(HIV-1) at 39° C. for 2 days has been documented to significantlydecrease viral production and reduce reverse transcriptase enzyme markeractivity 30 fold. In vitro hyperthermia of 42.0° C. for 1 hour, 4 daysapart selectively lowers HIV RNA loads in chronic (latent) infected Tlymphocytes. Hyperthermia of 42° C. for 3 hours combined with tumornecrosis factor has been published to selectively kill all acute andchronically infected HIV cells in tissue culture.

Use of whole body hyperthermia has been reported to cause regression ofKaposis' sarcoma, clear oral candidiasis, eliminate hepatitis C, causeremission of Varicella-zoster, increase weight gain and improve CD4lymphocytes counts in patients with acquired immunodeficiency syndrome(AIDS). Dramatic improvement with hyperthermia therapy has beendocumented in a patient infected with a debilitating Verruca vulgarisand HIV. The FDA has approved clinical trials involving hyperthermia forthe treatment of AIDS with a patented extracorporeal blood heatingmachine to induce whole body hyperthermia. The FDA has recently expandedthe extracorporeal heating machine trials to permit treatment of 40 HIVinfected patients.

Hyperthermia can augment cytotoxicity and reverse drug resistance tomany chemotherapeutic agents. Moreover, hyperthermia has also been shownto enhance the delivery of many novel cancer therapeutic agents, i.e.,monoclonal antibodies to neoplasms with resultant improvement inantitumor effect; enhance the delivery of gene therapy with use of viralvectors; and, augment drug delivery and antitumor effects when usingdrug containing liposomes. In addition to increasing the rate ofextravasation of liposomes from the vascular compartment by a factor of40-50, hyperthermia can also be used to selectively releasechemotherapeutic agents from liposomes designed to be thermosensitive.Thermosensitive liposomes are small vesicles composed of lipidphosphatidylcholine moieties constructed to contain and transport avariety of drugs. The liposomes are designed to remain stable in theblood and tissues at physiologic temperatures. When passing through anarea of heated tissue however, they dissolve and effectively releasetheir encapsulated contents. Thermosensitive liposomes are used toentrap and carry drugs whose systemic toxicity is desired to be limitedto a particular heated tumor, organ or tissue. Examples of drugs thathave been encapsulated into liposomes include methotrexate, doxorubicin,amphotericin B, cisplatin and others. Liposomes can be designed so as torelease their contents at pre-determined temperatures.

Hyperthermia has also been an effective solution for the treatment of avariety of heat labile toxin or poisonous envenomations. For example, aneasy treatment for Scorpaenidae and Siganidae envenomation is the localapplication of heat. The major poisonous component of this and manyother venoms from lionfish, weever fish, bullrout, sculpin, surgeonfish, scorpion fish, stonefish, butterfly cod, etc., is a very heatlabile, non-dialyzable protein. As opposed to the nuances of usingspecific anti-venom, emmersing the envenomated area or patient in hotwater, or applying other forms of hyperthermia, is a simple and prompttreatment.

Standard clinical methods of inducing hyperthermia are dependent on thedeposition of exogenous heat to that normally produced by themetabolism. All current deliberate and controlled methods of heatingrequire an external source of energy. Non-surgical methods of heatinginclude: hot air, ultrasound, microwaves, paraffin wax baths, hot waterblankets, radiant heat devices, high temperature hydrotherapy andcombinations thereof. Invasive means of inducing hyperthermia includesurgical insertion of various heating devices, infusion of heatedsolutions into the peritoneal cavity through catheters or heating theblood extracorporeally through a heat exchanger. The later method,developed by Parks et al., involves the surgical placement of a femoralarterio-venous shunt for the removal, heating and replacement of bloodto induce whole body hyperthermia. A more recent experimentalimprovement on this method has been the induction of whole bodyhyperthermia with veno-venous shunt perfusions. Several machines havebeen patented for extracorporeal heating of blood to induce hyperthermia(see U.S. Pat. Nos. 5,391,142 and 5,674,190).

Endogenous heating by creating fevers induced with toxins, pyrogens andmicroorganisms have been used in the past and have recently beenre-attempted. Heimlich has been reported to use Malaria therapy for thetreatment of Lyme disease, AIDS and malignancy. Pontiggia et al, treatedAIDS patients by combining fever, induced by parenteral injections of astreptococcal lysate preparations, with hyperthermia generated by aninfrared heating bed.

Another way that the prior art has dealt with inducing hyperthermia hasbeen by introducing micron size magnetic particles and subjecting themto either magnetic fields or hyperbaric oxygen (see U.S. Pat. No.4,569,836). This method was designed for the treatment of cancer basedon the belief that cancer cells would engulf the particles andconcentrate them intracellularly. A magnetic field would then be appliedto heat the particles and generate lethal hyperthermia within the cancercells. A modification of this technology is the use of magnetic cationicliposomes to induce intracellular hyperthermia. This technology wasbased on the observation that glioma cells have a greater affinity forpositively charged rather than ‘neutral’ magnetic lipsomes. A morerecent variation on this science has been developed in Germany using‘targeted’ magnetoliposomes. This methodology has been developed in anattempt to treat AIDS by using magnetic nanoparticles coupled to eitherCD4 lymphocyte or anti-gp120 HIV antibodies. The magnetic nanoparticlesare intended to selectively bind to either the HIV protein envelope orthe HIV infected cells and then be heated by external high-frequencyalternating magnetic fields.

Whether invasive or non-invasive, all current methods of inducinghyperthermia depend on an external energy source and cannot safelydeliver adequate power to result in therapeutic heating. Delivery ofheat to obtain the actual desired temperature to deep target tissues hasnot been possible because of the actual physics involved in thethermodynamic, conductive transfer of heat from the outside into thecell. Heating tissues deeper than five centimeters below the skin withmicrowave, radio frequency or ultrasound devices is difficult becauseenergy absorption is not uniform or focused. Radiant heat, hot water,molten wax and other methods cause excessive heating of subcutaneous fatwhich acts as a barrier to body heat gain. Common adverse effects ofsuch external heating methods include surface skin burns, blistering,ulcerations, secondary opportunistic infections and pain. Additionally,many tumors have high blood flow cooling which nullifies any potentialtherapeutic gain achievable through the use of such extracellular,systemic hyperthermia devices. Also, insufficient heating power prolongsthe induction time required to reach the actual therapeutic temperature.This promotes resistance to heat treatment through the development ofthe heat shock response and thermotolerance.

High frequency electromagnetic devices used to heat intracellularmagnetic particles invariably induce eddy currents within the bodymaking it difficult to provide uniform, controlled and safe heatingwithout toxic effects to normal cells. Further, not all tumors possesscharacteristics that cause them to selectively take up magneticparticles or have an affinity for positively charged magnetic liposomes.Also, magnetic cationic liposome particles are subject to variousneutralizing interactions with anions, giving them a short chargedhalf-life. Moreover, the complexity of using specific anti-HIVantibodies bound to electromagnetic particles also assumes anon-mutating HIV genome with stable antigenic determinants. To thecontrary, a high mutation rate in the HIV genome and it's proteinantigenic determinants is known to exist and is the main obstacle to thedevelopment of an effective vaccine. Such treatments therefore, do notselectively heat transformed cells without heating and injuring normalcells.

Extracorporeal blood heating methods require surgery and anesthesia.Further, as with all external heating methods, temperature variances andtoxic conductive thermogradients from the point of initial heating tothe target tissue cannot be avoided. By example, bone marrowtemperatures are consistently known to be 1°-2° C. below the averagebody core temperature achieved by extracorporeal blood hyperthermia.This is a major problem in systemic hyperthermic therapy since themarrow is a common repository of metastatic cancer cells and infectiousmicroorganisms. Therapeutic bone marrow temperatures are not achievabledue to the fact that the intermediate tissues between the blood and themarrow create a temperature gradient cooling the blood before it reachesthe bone marrow. Since efficacy and toxicity of hyperthermia depend onboth the actual temperature and duration of heating, delivering thedesired temperature-and-duration of heating (thermal dose) to the bonemarrow would require the blood and intermediate tissues to be heatedbeyond that which is safe for normal, healthy cells. A multicentreEuropean trial documented that only 14% of all protocols achieverequired target temperatures. Further, current extracorporeal heatingmethodology and equipment is labor intensive, time-consuming andexpensive.

Use of fever inducing agents such as live microorganisms, pyrogens andtoxin lysates is clinically uncontrollable, unpredictable orinsufficient as to both the degree and duration of temperature increase.

Further reasons why hyperthermia has not yet become more widely acceptedas a mode of therapy is because current heating machines are notcompatible with noninvasive temperature measurement technology.Measurement of the actual temperatures reached in target tissues iscritical for heating efficacy, i.e., determining the thermal dose.Recently, noninvasive thermometry with Magnetic Resonance Imaging (MRI),ultrasound backscatter, electrical impedance, electromagnetic adaptivefeedback and advanced, high-precision pixel infrared temperature imaginghave been developed. To use MRI or other equipment to monitor real timehyperthermia however, it is necessary to combine a hyperthermia devicewith an MRI unit. This has proven to be difficult and costly since eachdevice is functionally disturbed, if not damaged, by the presence of theother.

The exact molecular and cellular mechanism by which heat kills orinactivates tumor cells and microorganisms is unknown. Heat is anentropic agent and acts globally on every molecule constituting thecell. Heating is known to cause conformational changes in proteins,denature enzymes and affect cell membrane fluidity. By example, herpessimplex virus (type 1) thymidine kinase has a shortened half-life at 40°C. of only 30 minutes. The transforming gene product-enzyme of Roussarcoma virus (protein phosphatase), a critical protein for cellularregulation, is totally inactivated in 30 minutes at 41° C. Hyperthermiais known to increase the formation of oxygen free radicals, includingsuperoxide, hydroxyl, hydroperoxyl, hydrogen peroxide and lipidperoxides. These reactive oxygen species react indiscriminately andoxidize many organic molecules causing DNA damage, protein denaturation,lipid peroxidation and other destructive chain reactions. Acidmicroenvironments, known to exist in tumors and microorganisms with highrates of glycolysis (Embden-Meyerhof Pathway) and lactic acidproduction, favor protonation of the superoxide radical to form thehighly reactive and toxic hydroperoxyl radical. Thus, thermalsensitivity of many tumors increases with decreasing intracellular pH.As compared to normal cells, many malignant and virally transformedcells have a reduced total functional capacity to withstand the increaseflux of oxygen free radicals produced by hyperthermia.

On the intracellular level, moderate heating is known to activatephospholipase A₂, which increases the formation of pro-inflammatorymediators such as the leukotrienes, prostaglandins and eicosanoids. Heatalso increases release of intracellular calcium through the stimulationof phospholipase C. Calcium cycling across the mitochondrial membraneappears critical to the increased production of oxygen free radicals.Increased intracellular calcium also inhibits the mitochondrial,anti-apoptotic Bcl-2 protein and induces the production of heat shockproteins, mediating thermotolerance. Heat injury to the intracellulartubulin network, lysosomes, Golgi bodies, mitochondria, and control ofRNA splicing are some of the many known subcellular systems affected byheat. While the initial primary event leading to cell death byhyperthermia is unknown, a decrease in mitochondrial membrane potentialfollowed by uncoupling of oxidative phosphorylation and generation ofreactive oxygen species on the uncoupled respiratory chain are the firstbiochemical alterations detectable in cells irreversibly committed toapoptosis. The cytotoxic effect of hyperthermia is thus believed to becaused by numerous changes and complex damage to multiple vital cellfunctions. Those biochemicals altered by heat and essential to thefunction or viability of the cell are the pivotal targets of therapeuticheating.

The mode of hyperthermic cell injury is dependent on the severity of theheat stress, temperature and duration of heating. Moderate heating of39°-42° C. is used therapeutically and is known to promote programmedcell death through apoptosis, an active process of selectivelyeliminating heat sensitive cells without inflammation, bystander-celldeath or subsequent tissue fibrosis. Malignant and other transformedcells undergo apoptosis by suppression or activation of one or moregenes such as bcl-2, c-myc, p53, TRPM-2, RP-2, RP-8, raf, abl,APO-11FAS, ced-3, ced-4, ced-9, etc. Drugs (methotrexate, cisplatin,colchicine, etc.), hormones (glucocorticoids), cytokines (tumor necrosisfactor-alpha), radiation (free radicals) and hyperthermia can allinitiate apoptosis. Increasing the temperature or duration of heating,or both, leads to cell death via necrosis. This physical process ofindiscriminate cell killing is associated with inflammation and causessignificant injury to normal, healthy cells.

For purposes of systemic hyperthermia, apoptosis of target cells is thetherapy of choice. In the clinical setting it must be controlled underconditions of moderate heating so as to selectively differentiate andeliminate target cells with minimum toxicity to normal cells. Suchcontrolled conductive heating by external technologies is inherently notpossible. The thermal physical and thermophysiologic properties of cellsvary and are dependent on their thermal conductivity, specific heat,density and blood perfusion among the various organs and tissues. Basedon these properties, the actual temperatures at some of these sites areoften ‘partitioned’, independent of one another and do not represent themonitored, mean “core” temperature achieved during therapy.Additionally, it is well recognized that it is the actual intracellulartemperature increase, with it's associated internal physical andchemical changes, that is critical to the successful use of hyperthermiain exploiting the fundamental biochemical differences between normal andheat susceptible cells. Unfortunately, the initial cellular targets ofall extracorporeal heating methods are the cell membrane and it'sintegrated proteins. The cell's internal contents, includingmitochondria, compartmentalized enzymes, other organelles and anyintracellular pathogens, etc., are progressively heated in sequence bythermal conduction from the outside-in. Thus, to sufficiently heat theinterior of the cell, the external temperature must overcome thecellular and mitochondrial membranes, each composed of a lipid bilayerthat acts as an effective thermal barrier.

By necessity, therefore, prior art heating methods require high externaltemperatures to establish a sufficient gradient to overcome thenonisotropic and non-homogeneous conductive heat loss between internaltissues and the insulating barrier of the cellular and mitochondrialmembranes. For example, the Organetics PSI® (now First Circle MedicalInc.) device has to heat blood externally to 48° C. (118.4° F.) beforereturning it directly into the vascular system of the patient. Otherextracorporeal circuit perfusion devices need to achieve ex vivotemperatures of 49° C. (120.2° F.). Animal studies require temperaturesof 54° C. (129.1° F.) during the induction phase to achieve adequatetarget tissue temperatures. Safety in such prior art is thereforelimited by the incipient destruction of surrounding tissues at the sitesof the high temperature phases of heating. When lesser temperatures areattempted, effectiveness is compromised by either inadequatetemperatures or duration of heating or development of thermotolerance.As a result, only regional hyperthermia has been widely used clinicallyand only in combination with more traditional techniques such asradiation and chemotherapy. Presently, none of the known heatingtechnologies provide clinically safe and effective hyperthermia to treatsystemic or disseminated disease. In order for systemic hyperthermia tobecome more widely used clinically, current heating methods must alsoovercome the use of labor intensive, complex equipment, includinginvasive extracorporeal infusion and it's related toxicity problems tointerposed tissues. Further, new hyperthermic technology must becompatible with noninvasive, real time thermometry.

The present invention avoids the problems of heat toxicity, inadequatetarget tissue heating, excessive cost, surgery, anesthesia andincompatibility with noninvasive temperature measuring devices: problemsthat are inherent to all therapeutic methods that deliver heatextracellularly, from the outside-in. This invention is anintracellular, therefore, an intracorporeal heating system which hasadditional distinct advantages. First, the human body is biochemicallyand physiologically designed to tolerate higher temperatures when heatedfrom the inside-out as opposed from the outside-in. By example, incomparison to extracorporeal heating, which can safely generate amaximum body core temperature of 42° C. (107.6° F.), intracorporealhyperthermia caused by strenuous exercise induces physiologictemperatures of up to 45° C. (113.0° F.) in muscle and liver with bodycore temperatures of up to 44° C. (111.2° F.). Exertional heat strokepatients have survived rectal body temperatures as high as 46.5° C.(115.2° F.) without any permanent clinical sequela. While the criticalmaximum temperature humans can tolerate is unknown, physiologichyperthermic temperature induced under controlled conditions withadequate hydration have not shown any permanent untoward effects. Liverbiopsies from subjects with such temperatures have not shown anysignificant microscopic abnormalities. Second, since heating with thepresent invention is chemically induced from within the cell, the actualintracellular therapeutic temperature will be higher than the measuredcore temperatures. As a result, intracellular organelles, includingmitochondria, are heated at higher temperatures, undergo greateruncoupling and generate an increased flux of reactive oxygen species.Since oxygen free radicals, including superoxide, enhance and probablymediate the effects of hyperthermia, an improved therapeutic gain willbe obtained at lower body core temperatures. Further, it is known thatfor each 0.5 degree Celsius increase in body temperature the metabolicrate and oxygen consumption increase 7%. Such an increase will assistheating the body in itself. Third, safety and control of temperatureswith the present invention is far superior to that of exogenous methods.The body is naturally designed to dissipate heat from the inside-out.This is evident from the fact that a temperature gradient of 3.5°-4.5°C. exists between the visceral core and the skin. This gradientrepresents the transfer of heat from regions of high temperature toregions of low temperature, with ultimate heat loss from the skin to theenvironment through conduction, convection, radiation and sweat inducedevaporation. The margin of safety and control represented by the‘feedback gain’ of this intact physiologic heat dissipating system isextremely high, approximating 27-33. This rate of cooling can balance aninflux of heat in a naked human body in a dry room at about 120° C.(248.0° F.). Thus, the human heat flow system permits the body to riditself of excess endogenous heat very quickly and effectively. As aresult, there is a wide margin of safety in case the target temperatureis exceeded. In contrast, exogenous heating contravenes the naturalphysiologic flow of heat and its dissipating mechanisms. The naturalheat dissipating mechanisms are overwhelmed and compromised. Control andsafety over hyperthermia induced by extracellular means is thus fragile,with little room for error.

SUMMARY OF THE INVENTION

The present invention encompasses a composition and method usingmitochondrial uncoupling agents, especially DNP, DNP with free radicalproducing drugs, DNP with liposomes, DNP conjugated to free radicalformers, and DNP with other therapeutic pharmaceutical agents which areactivated intracellularly by heat or reaction with mitochondrialelectrons or free radicals to cause release of active medications forthe treatment of cancer, HIV, other viruses, parasites, bacteria, fungiand other diseases. While not being bound by theory, it is submittedthat the use of mitochondrial uncoupling agents, to increaseintracellular heat and free radicals, as treatment for non-relatedcancers, viruses and other pathogens presupposes that the mechanism ofaction is non-specific for enzymes and receptors but is specific forinterference with cellular and pathogen viability and induction ofprogrammed cell death. The degree of intracellular heating, free radicalformation, whole body hyperthermia and release of active drug moleculesis controlled by the dose of DNP. Based on the quantity of oxygenconsumed, the dose of DNP is adjusted to achieve the desired degree ofhyperthermia. Safety and effectiveness is further controlled bymanipulating metabolic rates of target tissues, duration of treatmentand permissiveness of body cooling. In accordance with the presentinvention, intracellular, mitochondrial heat is generated by the use ofDNP, other uncouplers, their conjugates, either alone or in combinationwith other drugs for the treatment of thermosensitive cancers such asnon-Hodgkins lymphoma, prostate carcinoma, glioblastoma multiforme,Kaposi's sarcoma, etc; bacteria such as Borrelia burgdorferi,Mycobacterium leprae, Treponema pallidum, etc.; viruses such as HIV,hepatitis C, herpes viruses, papillomavirus, etc.; fungi such asCandida, Sporothrix schenkii, Histoplasma, Paracoccidiodes, Aspergillus,etc.; and, parasites such as Leishmania, malaria, acanthomoeba,cestodes, etc. 2,4-dinitrophenol was selected as the uncoupler of choicebecause it can be used at relatively high concentrations, permittinguniform distribution in organs and tissues. This invention alsoencompasses the use of DNP to selectively augment energy metabolism andheat production in inchoate malignant tumors for the purpose ofincreasing sensitivity of diagnostic positron emission tomography,temperature-sensitive magnetic resonance, and high-precision pixeltemperature infrared imaging in differentiating normal from aberrantcell metabolisms. An additional object of the invention is the use ofDNP to increase transcription of heat shock proteins, especially HSP 72,as a form of cellular pre-conditioning to decrease post-angioplastyrestenosis, increase successful outcome of other surgeries, andfacilitate antigen processing and presentation of immunogenicdeterminants on infectious agents, virally transformed cells and tumorsso as to increase the natural or biologically activated immunologicalresponse.

In accordance with another aspect of the present invention, controlledthermogenesis with DNP is combined with other agents used to treatinfectious, malignancy and other diseases. Examples of other agentsinclude antifungal, antiviral, antibacterial, antiparasitic andantineoplastic drugs. Such drugs, including angiogenesis inhibitors andradiation have increased synergistic or additive activity when combinedwith hyperthermia in the treatment of cancer.

The method can be used for enhancing the sensitivity of positronemission tomography, nuclear magnetic resonance spectroscopy andinfrared thermography in the diagnosis and monitoring of treatment ofvarious diseases, including cancer. Similarly, the method can be usedfor enhancing the identification of unstable “hot” coronary and carotidartery plaques predisposed to rupture or undergo thrombosis. Suchdiagnostic and treatment monitoring methodology is based on the factthat most tumors have higher metabolic rates and generate more heat thannormal tissues. Likewise, unstable atherosclerotic plaques are presumedto rupture because they have a dense infiltration of macrophages whichhave high metabolic rates and generate excessive enzymes and heat,causing the plaque to degrade and loosen. In both instances, controlleddoses of DNP or other uncouplers can further increase metabolic ratesand heat production to increase diagnostic sensitivity. Controlledheating with DNP and fibrinolytic recombinant tissue-type plasminogenactivators can also be used therapeutically to accelerate fibrinolysisof clotted arteries.

In another aspect of the invention, DNP is administered in controlledand timed dosages to provide physiologic stress, “chemical exercise”, soas to induce synthesis of autologous heat shock proteins (HSPs).Intracellular heat exposure associated with autologous HSP induction hasa significant cytoprotective effect against ischemia and cellular traumaand acts as a form of cellular thermal preconditioning in patients aboutto undergo surgery. Induction of HSPs by DNP in patients some 8 to 24hours prior to angioplasty, coronary bypass surgery, organtransplantation and other forms of high risk surgery, would provide forimproved clinical outcome with decreased post-angioplasty intimalthickening or restenosis, increased myocardial protection frominfarction, improved musculocutaneous flap survival in plasticreconstruction and reduced ischemia/reperfusion injury in organtransplantation cases.

Another aspect of the invention provides for controlled dosages of DNPto induce long duration (6 to 8 hour), mild whole body hyperthermia(39.0 to 40.0° C.) to afford maximum expression of immunogenic HSPs orpeptides associated with HSPs. The antigenic properties of HSPs andHSP-peptide complexes, induced by DNP in infectious agents, especiallythose located intracellularly, or on tumors can be exploited to enhancethe immune response. This aspect of the present invention provides aprocess for modulating the immune system of a patient with othertherapies, comprising the steps of: (1) increasing the expression ofHSPs by the process described above, and (2) administering humanizedmonoclonal or polyclonal antibodies, or (3) administering recombinantcytokines, lymphokines, interferons, etc., or (4) administering standardanti-infectious or anti-neoplastic therapy.

Additional objects and advantages of the invention will be set forth inpart in the description of drawings that follows, and in part, will beobvious from the description, or may be learned by practice of theinvention. The objects and advantages can be realized and obtained bymeans of the uses and compositions particularly pointed out in thedetailed description of the preferred embodiments and in the appendedclaims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows features of glycolysis with formation of pyruvic acid andrelease of energy as heat.

FIG. 2 depicts the conversion of pyruvic acid into acetyl CoA and the 2carbon fragments entering the TCA cycle.

FIG. 3 shows the transfer of electrons down the electron transport chainduring the process of oxidative phosphorylation

FIG. 4 shows oxidative phosphorylation as a coupling of two distinctprocesses, oxidation of reducing equivalents and formation of ATP. Bothprocesses are “coupled” by an electrochemical membrane potential createdby electrons passing down the electron transport chain.

FIG. 5 shows the process of chemiosmosis. Electrons passing down theelectron transport chain create energy to pump H⁺ outside the innermitochondrial membrane. This process creates a protonmotive force thatcauses formation of ATP by protons re-entering the membrane throughATP-synthase.

FIG. 6 depicts the uncoupling of oxidative phosphorylation throughinjury of the inner mitochondrial membrane. FIG. 6( a) shows howoxidative phosphorylation is uncoupled by DNP in intact and uninjuredmitochondrial membranes.

FIG. 7 shows the initial formation of superoxide radicals by theunivalent reduction of oxygen in the electron transport chain. FIG. 7(a) depicts the formation of hydrogen peroxide and hydroxyl radicalsthrough the Haber-Weiss Reaction. FIG. 7( b) shows an overview ofmitochondrial oxygen utilization and free radical formation.

FIG. 8( a) depicts the effects of heating on mitochondrial uncouplingand FIG. 8( b) shows the correlation of uncoupling to superoxide freeradical formation.

FIG. 9 depicts the increased formation of oxygen free radicals aftercessation of DNP uncoupling and normalization of oxygen consumption.

FIG. 10 shows the global intracellular effects of DNP, including thedominant foci of increased heat generation.

FIG. 11 shows the relative potencies of various uncouplers.

FIG. 11( a) shows the effect of body temperature on metabolic rate.

FIG. 12 shows six of the Hottest organs in the human body and theirrelative blood flow.

FIG. 13 shows the effect of successive doses of 2,4-DNP on oxygenconsumption.

FIG. 14 shows a typical DNP induced hyperthermia patient monitored flowchart.

FIG. 15 shows a monitored patient flow chart after successive infusionsof DNP and glucagon for treatment of parasitic disease of the liver.

FIG. 16 shows killing of chronically HIV infected HUT-78 cells withvarying concentrations of DNP.

FIG. 17 shows a patient flow chart after infusion of norepinephrine andsuccessive intravenous doses of DNP for treatment of HIV disease.

FIG. 18 shows a monitored patient flow chart after successive infusionof DNP for treatment of Lyme disease.

FIG. 19 shows a monitored patient flow chart using an alpha-1 adrenergicagonist with DNP to induce hyperthermia in a patient with disseminatedcancer.

FIG. 20 shows survival studies of tumor growth-regressed animals treatedwith DNP and a thermosensitive liposome encapsulated drug.

FIG. 21 parts, 1, 2 and 3 shows the protective effects of DNPpretreatment on arterial catheter balloon induced injury.

FIG. 22 shows the protective effects of DNP pretreatment on survivalafter prolonged hepatic eschemic induced by Pringle's maneuver.

FIG. 23 shows the improved effect of musculocutaneous flap skin survivalafter DNP pretreatment.

FIG. 24 shows the effects of oral DNP on oxygen consumption prior to apatient undergoing a PET scan.

FIG. 25 shows a monitored DNP flow chart with incremental increases inoxygen consumption prior to a patient undergoing diagnosticthermography.

FIG. 26 shows a monitored patient flow chart using dinitrophenol andmethylene blue for the treatment of prostate carcinoma.

FIG. 27 shows biochemical and clinical response of androgen-independentprostatic carcinoma to dinitrophenol and methylene blue treatment.

FIG. 28 shows a monitored patient flow chart using interferon-alpha anddinitrophenol for the treatment of chronic hepatitis C infection.

FIG. 29 shows the effects of dinitrophenol and interferon-alphatreatment on liver enzymes and hepatitis C viral loads.

FIG. 30 shows an exemplary method of synthesis of novel2,4-dinitrophenol conjugates and derivatives.

FIG. 31 shows synthesis of an expanded combinatorial library ofuncoupling agents.

DETAILED DESCRIPTION OF THE INVENTION

Electron transferring, transporting and energy converting elements areubiquitous and are necessary for life. All eukaryotic and prokaryoticorganisms depend on electron transferring and transporting elements suchas metal containing hemes and nonmetal moieties such as flavins andadenine nucleotides. These biochemical entities convert the energystored in chemical bonds of foodstuffs into cellular and organellemembrane potentials, high energy containing molecules such as adenosinetriphosphate (ATP), creatinine phosphate, and other forms of chemicalenergy needed to maintain the highly negative entropic state of life.

The most common form of biologic energy is adenosine triphosphate (ATP).ATP is produced either anaerobically through the Embden-Myerhoff Pathway(glycolysis) or through oxidative phosphorylation. The latter, an oxygendependent chemical energy conversion process, is generally associatedwith the Tricarboxylic Acid Cycle [(TCA), Krebs Cycle or Citric AcidCycle]. The TCA cycle links the products of glycolysis to a multi-enzymecoupled series of electron carriers called an electron transport chain(ETS). The electron transport chain is coupled to production of ATP. Theentire TCA cycle and oxidative phosphorylation process is located inintracellular organelles known as mitochondria.

While release of energy from foodstuffs can come about through a varietyof biochemical means, the most important means by which energy releaseis initiated is by splitting glucose into two molecules of pyruvic acid.This occurs through the non-oxygen dependent process of glycolysis in aseries of ten chemical steps depicted in FIG. 1. The overall efficiencyof trapping energy in the form of ATP through this anaerobic process is43%. The remaining released energy (57%) is discharged in the form ofheat.

Pyruvic acid molecules derived from glucose, as well as end products offat and protein breakdown, are transported into the mitochondrial matrixwere they are converted into 2 carbon fragments of acetylcoenzyme A,FIG. 2. As depicted, these acetyl fragments enter the TCA cycle weretheir hydrogen atoms are removed and released as either hydrogen ions(H⁺) or combined with nicotinamide and flavin adenine dinucleotides(NAD⁺ and FADH) to produce large quantities of usable reducingequivalents (NADH and FADH₂). The carbon skeleton is converted to carbondioxide (CO₂) which becomes dissolved in body fluids. Ultimately thedissolved CO₂ is transported to the lungs and expired from the body. Asnoted in FIG. 2, the flux of reactants in the TCA cycle is always in thesame direction because NADH and FADH₂ is constantly removed as hydrogenis oxidized by the mitochondrial electron transport chain.

It is the electron transport chain that provides approximately 90% ofthe total ATP formed by glucose catabolism. During this process, knownas oxidative phosphorylation, hydrogen atoms that were released duringglycolysis, the TCA cycle, and converted to NADH and FADH₂, are oxidizedby a series of enzymatic redox complexes (electron transport chain)located in the inner mitochondrial membrane, FIG. 3. Energy released inthese steps is captured by a chemiosmotic mechanism that is dependent onthe ultimate reduction of O₂ to form H₂O. As depicted in FIG. 4,oxidative phosphorylation is two distinct processes: (1) oxidation ofNADH and FADH₂; and, (2) formation of ATP. Both processes areinterdependent or “coupled” by a high energy linked proton (H⁺, pH)gradient and membrane potential across the inner mitochondrial membraneprovided by electrons as they pass through the electron transport chain.Energy released by the electrons pumps hydrogen ions (H⁺) from the innermatrix of the mitochondrion into the outer inter-membrane space, FIG. 5.This process is known as chemiosmosis and creates a high concentrationof H⁺ outside the inner mitochondrial membrane and a powerful negativeelectrical potential in the inner matrix. This transmembrane protongradient (protonmotive force) causes hydrogen ions to flow back into themitochondrial matrix through an integral membrane protein (ATP synthase)to form ATP from ADP and free ionic phosphate. The efficiency ofoxidative phosphorylation in capturing energy as ATP is about 69%. Theremaining (31%) liberated energy is dissipated as heat. The overallefficiency of energy transfer to ATP from glucose via glycolysis, theTCA cycle and oxidative phosphorylation is 66% with about 34% of theenergy being released as heat.

Heat is continually produced by the body as a byproduct of metabolismand eventually all energy expended by the body is converted to heat. Ona thermodynamic basis, total body heat production is the algebraic sumof the enthalpy changes of all biologic processes in the body. Thepathways are irrelevant, even though in the body oxidation involvesnumerous enzyme catalyzed reactions taking place at 37° C.Biochemically, approximately 95% of all the oxygen (O₂) consumed is usedby mitochondria to stoichiometrically couple oxygen reduction to ATP andheat production via oxidative phosphorylation. The rate of O₂consumption (VO₂) can be measured by indirect calorimetry and thusrelated to body heat production. Although this method does not includeanaerobic processes such as glycolysis, indirect calorimetry is in closeagreement with direct body heat measurements and it is generallyaccepted that 1 liter of VO₂ generates 4.825 Kcal (kilocalorie ofenergy), 5/li^(ths) of which can be detected as heat.

In human adults, increased VO₂ and endogenous heat production can occurvia muscular (work or shivering) and/or chemical [(cathecholamines,thyroid, etc.) non-shivering] thermogenesis. Whereas muscular activitycan increase heat production 4-10 fold, non-shivering thermogenesis canonly increase heat production by a maximum of 15%. However, oxygenconsumption and non-shivering thermogenesis can dramatically increasewhen even mild injury to the inner mitochondrial membrane occurs so thatit is no longer intact and protons leak or reenter the mitochondrion,uncoupled to ATP synthesis. Heating, endotoxin, osmotic imbalance, etc.,can cause such injury, i.e., loss of coupling, with resultingrespiration and ATP metabolism proceeding independently andmaximally—respiration forward, phosphorylation in reverse. FIG. 6compares normal coupled respiration and ATP formation to that whichoccurs when there has been injury to the inner mitochondrial membrane.The increased reduction of oxygen results in increased heat production.

Additionally, certain chemicals, including biologicals, can selectivelyincrease the transport of protons across uninjured, intact innermitochondrial membranes and dramatically increase VO₂ and heatproduction. These compounds dissipate the electrochemical-protonmotivetransmembrane potential of mitochondria and uncouple the electrontransport chain from ATP synthesis. FIG. 6( a) depicts one suchuncoupling agent, DNP, cycling protons across an intact mitochondrialmembrane. DNP and other uncouplers permit each of the two distinctprocesses involved in oxidative phosphorylation to “unlink” and increasetheir rates according to their own separate kinetic and thermodynamicsignals, FIG. 6( b). Uncouplers increase respiratory rates, electrontransport, VO₂, heat production and increased utilization of foodstuffsubstrates through glycolysis and the TCA cycle. Controlled doses of anuncoupler will increase O₂ consumption and heat production with minimalor no decrease in ATP levels because of intracellular equilibrium shiftsin creatinine phosphate, oxidative phosphorylation reactants andincreased production of ATP through the anaerobic, glycolytic pathway.Excess or toxic doses of virtually all uncouplers however, will producesecondary untoward effects, including decreased respiration, decreasedheat production and eventual cellular death.

In addition to heat being a byproduct of oxidative phosphorylation,reactive oxygen species are also continuously produced by themitochondrial electron transport chain. Free radicals of oxygen areproduced during aerobic oxidation as electrons are transported by theelectron carriers to ultimately reduce O₂ to H₂O. As depicted in FIG. 7,superoxide (O₂ ⁻) radicals are generated by leaked electrons through theunivalent reduction of oxygen. FIG. 7( a) shows that superoxidedismutase then converts the superoxide radical to hydrogen peroxide.Additional hydrogen peroxide (H₂O₂) and hydroxyl (OH⁻) radicals areformed through the Haber-Weiss Reaction, the hydroxyl radical being themost reactive species, reacting with any biologic moiety instantly. FIG.7( b) depicts the overall scheme of oxygen metabolism and free radicalformation at the level of the mitochondrion.

As mitochondria become progressively heated, uncoupling occurs withincreased flux of oxygen free radicals. The effects of heat onmitochondrial uncoupling and superoxide radical generation are depictedin FIG. 8( a) and FIG. 8( b). A linear correlation of 0.98 (P<0.01) isobtained for the relationship between percent uncoupling and percentsuperoxide generation. Similar to exercise increased body temperatureand VO₂, hyperthermia induced by uncoupling agents appears to inhibitelectron transport at the level of cytochrome c in the redox chain.Normal rat liver, infused with DNP, increases formation of reactiveoxygen species threefold upon cessation of uncoupling, FIG. 9.

Generally, uncouplers are agents that are hydrophobic ionophores whichbind protons and traverse biologic membranes to dissipate transmembraneproton (pH) and membrane potential gradients (ΔΨ, Delta Psim). In sodoing, uncouplers increase the rate of metabolism (substrateutilization) in intact animals and isolated tissues by increasing therate of oxygen reduction through increased availability of protons. 0₂consumption is increased and remains rapid as long as the mitochondrialrespiratory (electron transport) chain attempts to overcome the effectsof the uncoupler to maintain a pH gradient. Energy is still used to pumpprotons across the mitochondrial membrane, but the protons are carriedback across the membrane by the uncoupler as depicted in FIG. 6( a).This creates a futile cycle and energy is released as heat. Thischemical heat releasing process is comparable to heating that occurswhen an electrical wire is “short circuited”. Depending on the degree ofexternal body heat dissipation, body temperature rises some 30 to 60minutes after the increase in 0₂ consumption. Onset of action is rapidafter an intravenous injection of an uncoupler. Depending on theintravenous dosage, human oxygen consumption is increased in about 15-20minutes and the intracellular heat production is increasedproportionately. Metabolic rates as high as 10 times normal have beenreported. Persistent increases in the metabolic rate can continue aslong as 12 to 36 hours because of the long hydrophobic half-life ofuncouplers in tissues. Temperature increases can be seen within 10 to 15minutes in subjects whose heat dissipation mechanisms have beencompromised. Heretofore, hyperthermia induced by uncoupling compoundshas not been reported to have any therapeutic application.

While there are three general classes of uncoupling agents, eachcontaining specific uncouplers of oxidative phosphorylation, the presentinvention utilizes 2,4-dinitrophenol (DNP) as the preferred embodiment.This is because DNP has been extensively studied. DNP was commonly usedin food dyes in the late 1800's and in the munitions industry of WorldWar I. Rapid increased respiration and hyperthermia, up to 49° C., wasnoted in man and animals that were accidentally intoxicated. Suchdramatic physiologic effects by the dinitro-aromatic dyes, especiallyDNP, caused them to be inextricably tied to early and later modernstudies of metabolism and bioenergetics. In the 1930's DNP wasintroduced into clinical medicine for the purpose weight loss. It was,however, sold as an over the counter secret nostrum and seriouslymisused. Had its long half-life in tissues been recognized and physiciansupervision implemented, it might have become an accepted drug. DNP hasbeen reported in countless, different enzyme, cellular and metabolicstudies. Review of such vast published studies have documented DNP'svery specific mechanism of action as a proton ionophore, with all othereffects a direct pharmacologic extension thereof. DNP is not mutagenicby the Ames and modified Ames tests; it has not been found to becarcinogenic or teratogenic; and, DNP blood plasma levels can easily bedetermined. DNP can be used at pharmacologic doses that achievetherapeutic concentrations in tissues. Further, DNP is stable,inexpensive and commercially available in reagent grade purity. It isunderstood however, that other uncouplers and combinations of otheruncouplers with other drugs, hormones, cytokines and radiation canpotentially be used under appropriate clinical settings and dosages toinduce intracellular hyperthermia and promote additive or synergisticeffects.

FIG. 10 shows the overall intracellular mechanism of action of DNP (andother uncouplers). Intracellular foci of increased heat and oxygen freeradical flux are highlighted. Circled numbers in the figure indicateboth direct and indirect effects of DNP: circled 1 and 2 effects showsthat upon its intercalation into the inner mitochondrial membrane, DNPshuttles H⁺ (hydrogen ions) across the membrane [see FIG. 6( a)]—thisshort circuits (de-energizes) the proton gradient established by the H⁺pumping action of the mitochondrial electron transport system (see FIG.5). As a consequence, the inner mitochondrial membrane potential islowered from −180 to −145 mV. Circled 3, 4, 5 and 6 effects shows thatnormal oxygen consumption and flux of NADH and FADH₂ (reducingequivalents) through the electron transport system is coupled to H⁺re-entry via mitochondrial availability of ADP for re-synthesis of ATP(see FIG. 4). By freely returning protons into the mitochondrial matrixwithout concomitant dependency on ADP to ATP reformation, DNP increasesoxygen consumption proportionately to the degree of uncoupling. The rateof oxygen consumption remains linked however, to the flux of electronsprovided by NADH and FADH₂ through the electron transport chain [seeFIG. 6( a)]. NADH and FADH₂ utilization (re-oxidation) is concomitantlyincreased. Circled 7, 8, 9, and 10 effects show that oxygen use andelectron transfer proceed at increasing rates to accelerate protonpumping against the added hydrogen ion load introduced by DNP. As aresult, NADH and FADH₂ is continually depleted by re-oxidation to NAD⁺and FAD⁺⁺. The high “oxidation pressure” of NAD⁺ and FAD⁺⁺ increasessubstrate oxidation and flux of 2 carbon segments through thetricarboxylic acid cycle (TCA). Augmented acetyl-CoA consumption in turnis maintained by an increased rate of glycolysis by depletion ofpyruvate. If oxygen delivery is inadequate, or the dose of DNPexcessive, the concentration of reduced NADH increases, pyruvateoxidation through acetyl-CoA and the TCA cycle is inhibited and lacticacid will accumulate. Lactate is also overproduced when cellular hypoxiais not present per se but glycolysis exceeds pyruvate oxidation. Suchintracellular lactic acidosis exists in neoplastic cells, when there islack of insulin, when fructose is infused and in other conditions or useof drugs which augment glycolysis and/or inhibit the mitochondrialelectron transport system. While it is understood that the intracellularheat generated by DNP is the algebraic sum of the enthalpy changes fromall the metabolic processes within the cell, effects circled as 11, 12and 13 depict the most significant intracellular foci of heat generatedby DNP. Intracellular and total body hyperthermia results when DNPreleases energy at a rate faster than it can be dissipated. Heat isgenerated mainly at the inner mitochondrial membrane (electron transportsystem), the TCA cycle and sites of cytoplasmic glycolysis. InitiallyDNP generates heat at the inner mitochondrial membrane by discharging aportion of the energy stored in its electrochemical gradient.Operationally, such heat is from the “chemical short circuit” created byDNP shuttling protons to the negative (matrix) side of the polarizedinner mitochondrial membrane [see FIG. 6( a)]. By usurping controlledproton re-entry and energy capture as ATP from availability of ADPthrough ATP-synthase, DNP causes NADH and FADH₂ (higher concentrationsof NAD⁺ and FAD⁺⁺) reoxidation to occur at rates much higher thannecessary for oxidative phosphorylation. This causes an increased fallof electrons through the electron transport chain with rapid reductionof oxygen to water (see FIG. 3). The resultant energy is released asheat within the mitochondrial membrane. The rate of heat production fromthe TCA cycle is increased as it operates at a higher flux to maintaindepleting amounts of reduced NADH and FADH₂ used to reduce molecularoxygen. Flux of acetyl-CoA and all metabolites through the TCA cycle(see FIG. 2) is increased by activation of enzymes which sequentiallydegrade the hydrogen containing two carbon fragments to CO₂, NADH, FADH₂and heat.

Glycolysis and its associated heat production in the cytoplasm is alsoincreased by DNP. Glycolytic activity is increased by reducedconcentration ratios of ATP to ADP, activating puruvate dehydrogenaseand phosphofructokinase respectively (see FIG. 1). These enzymesincrease the rate of glucose catabolism to pyruvate and its conversionto acetyl-CoA for entry into the TCA cycle. Glycolysis is very “energyinefficient” in making up the energy equilibrium shortfall created byDNP. Uncaptured energy from the glycolytic exergonic reactionsaccelerated by DNP is released as heat in the cytoplasm. DNP stimulatedanaerobic heat production through glycolysis can oftentimes be greaterthan that produced by the mitochondria. By example, many tumors andnormal fibroblasts treated with DNP increase heat production by 83%,with only a 36% increase in oxygen consumption. Glycolysis is known tocontribute greater than 62% of the total heat produced by humanlymphocytes. Circled effect 14 shows that the mitochondrial electrontransport chain normally produces reactive oxygen species through theunivalent reduction of oxygen [see FIGS. 7, 7(a) & 7(b)]. Underphysiologic conditions, 2 to 4% of mitochondrial oxygen is converted tosuperoxide. DNP induced partial uncoupling and mitochondrial heatingincreases reactive oxygen species production manifold. Cytochromeoxidase and reductase is known to be inhibited by heating of theelectron transport system. As a result, heated mitochondrial membranesproduce increased amount of oxygen free radicals when DNP induceduncoupling is stopped and oxygen consumption is normalized (see FIG. 9).Reactive oxygen species act in synergy with heat to alter proteins,induce membrane changes and initiate apoptosis in susceptible cells.Circled effects 15 and 16 shows the effects of DNP on intracellularcalcium homeostasis. Normally calcium is stored in the mitochondrialmatrix, being pumped by the energized mitochondrial membrane. By DNPdirectly de-energizing mitochondria, and indirectly inducing membraneheating and prooxidant stress, inner mitochondrial membrane permeabilityis non-specifically increased with calcium efflux and cycling. Thisactivates intramitochondrial dehydrogenses to produce more reducingequivalents in the form of NADH and FADH₂ to match increased energydemands. Heat production is increased as a byproduct from the augmentedTCA cycle. Other known uncouplers that are considered to be “classic”,in the same category and act as DNP include clofazimine, albendazole,cambendazole, oxibendazole, triclabendazole (TCZ),6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole and theirsulfoxide and sulfone metabolites, thiobendazole, rafoxanide, bithionol,niclosamide, eutypine, various lichen acids (hydroxybenzoic acids) suchas (+)usnic acid, vulpinic acid and atranorin,2′,5-dichloro-3-t-butyl-4′-nitrosalicylanilide (S-13),3,4′,5-trichlorosalicylanilide (DCC), platanetin,2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799,AU-1421,3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradecin-1,7(8H)-dione(zearalenone), N,N¹-bis-(4-trifluoromethylphenyl)-urea, resorcylic acidlactones and their derivatives,3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847),2,2,-bis(hexafluoroacetonyl)acetone, triphenyl boron, carbonylcyanide4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA), carbonylcyanide 3-chlorophenylhydrazone (ClCCP), 1,3,6,8-tetranitrocarbazole,tetrachlorobenzotriazole, 4-iso-octyl-2,6-dinitrophenol(Octyl-DNP),4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxalbisguanylhydrazone), pentachlorophenol (PCP),5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI),N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid),4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol,2-azido-4-nitrophenol, 5-nitrobenzotriazole,5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole,methyl-o-phenylhydrazone, N-phenylanthranilic acid,N-(3-nitrophenyl)anthranilic acid, N-(2,3-dimethylphenyl)anthranilicacid, mefenamic acid, diflunisal, flufenamix acid,N-(3-chlorophenyl)anthranilic acid, carbonyl cyanide4-trifluoromethoxyphenylhydrazone (FCCP), SR-4233 (Tirapazamine),atovaquone, carbonyl cyanide4-(6′-methyl-2′-benzothiazyl)-phenylhydrazone (BT-CCP), ellipticine,olivacine, ellipticinium, isoellipticine and related isomers,methyl-O-phenylhydrazonocyanoaceticacid,methyl-O-(3-chlorophenylhydrazono)cyanoacetic acid,2-(3′-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic acid,2-(2′,4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile,relanium, melipramine, and other diverse chemical entities includingunsaturated fatty acids (up to C₁₄ optimum), sulflaramid and itsmetabolite perfluorooctane sulfonamide (DESFA), perfluorooctanoate,clofibrate, Wy-14, 643, ciprofibrate, and fluoroalcohols. Additionalunnamed classic uncouplers can include any analog which generally has aweakly acidic, removable proton and an electron withdrawing, lipophilicmolecular body that is capable of charge delocalization. Hydrophobicityand capacity to exchange proton equivalents are integral features ofclassic DNP types of uncouplers.

A second class of uncouplers are ionophorous antibiotics. Thesemolecules uncouple oxidative phosphorylation by inducing cation or anioninflux across the mitochondrial membranes and diffusing back in aprotonated form. As a result, chemical futile cycling ensues toreestablish the initial membrane potential. Liberated energy isdissipated as heat. Examples of ionophores that shuttle potassium ions(K⁺) across membranes includes the antibiotics gramicidin, nigericin,tyrothricin, tyrocidin, and valinomycin. Nystatin shuttle sodium ions.The calcium ionophore, compound A23187, is a lipid soluble ionophorewhich mediates the electroneutral exchange of divalent cations forprotons. Alamethicins, harzianin HA V, saturnisporin SA IV, zervamicins,magainin, cecropins, melittin, hypelcins, suzukacillins, monensins,trichotoxins, antiamoebins, crystal violet, cyanine dyes, cadmium ion,trichosporin-B and their derivatives are examples of uncouplingionophores that depend on shuttling inorganic phospate (PO₄ ⁼) acrossthe mitochondrial membrane.

A third class of uncouplers is a group of heterogeneous compounds thatdissipate the proton gradient by attaching or interacting with specificproteins in the inner mitchondrial membrane. Examples of such compoundsinclude desaspidin, ionized calcium (Ca⁺⁺), uncoupling proteins such asUCPI-1, UCP-2, UCP-3, PUMP (Plant Uncoupling Mitochondrial Protein)histones, polylysines, and A206668-a protein antibiotic that ties upphosphoryl-transfer proteins. Examples and a potency comparison of a fewuncouplers are depicted in FIG. 11.

Various conjugates, adducts, analogs and derivatives of the abovementioned agents can be formulated and synthesized to enhanceintracellular uncoupling and heat production. Further, various covalentcompounds of uncouplers may be synthesized as prodrugs, which upon,redox or reaction with free radicals within the cell will becomeactivated to induce uncoupling, heat production and free radicalcycling. Such derivatives and formulations may be desirable in thetreatment of many tumors with higher mitochondrial membrane potentialsand increased total bioreductive capacity. Uncoupling-free radicalprodrug compounds may thus exert greater selective killing oftransformed cells by undergoing a higher flux of reduction or electronacceptance in tumor cells. In this regard, the contents of U.S. Pat. No.5,428,163 and the published methods of C-Alkylation of phenols and theirderivatives by Hudgens, T. L. and Turnbull, K. D. are herebyincorporated by reference

From a physico-chemical and thermodynamic standpoint, the amount of heatproduced by uncoupling is proportional to the density and rate of fluxof electrons through the mitochondrial electron transport chains. Suchelectron flux is initially reflected by the magnitude of theelectrochemical proton gradient across the inner mitochondrial membrane.Those cells, tissues, organs and organisms that are metabolically moreactive will generally have an increased membrane potential and willrespond with a greater amount of heat production for a given dose andtype of uncoupler. FIG. 12 lists the six most “hottest” organs in thehuman body along with their rates of blood flow and rates of heatproduction. The actual amount of intracellular hyperthermia produced byan uncoupler is dependent on the uncoupler dose, its relative potencyand availability of substrate such as glucose, glutamine, fatty acids orother substances that produce NADH or FADH₂. Oxygen and magnitude of themitochondrial proton electrochemical gradient (ΔμH⁺) are additionalfactors that determine the amount of heat that can potentially bereleased by an uncoupler. Among all the constituents, ΔμH⁺ is the mostclinically important. ΔμH⁺ is composed of the transmitochondrialmembrane potential [ΔΨ (charge difference)] and pH gradient [Δ pH (H⁺concentration difference)], ΔμH⁺=−2.3RTΔpH, where, F=Faraday Constant,R=Gas Constant, and T=degrees Kelvin. Thus, ΔμH⁺ represents thepotential amount of heat that can be liberated by an uncoupler when 1mole of H⁺ is dissipated through the inner mitochondrial membrane. Thispotential heat energy is normally expressed in units of millivolts (mV)and is called the protonmotive force, Δp=ΔμH⁺/F=ΔΨ−2.3(RT/F)ΔpH. Invivo, ΔpH is generally 1 unit or less so that 75% or more of the totalΔp is comprised of ΔΨ. Consequently, the intracellular heat produced byan uncoupler can be estimated by the mitochondrial membrane potential(ΔΨ) alone.

Knowing the ΔΨ is of practical importance because biopsy specimens maybe incubated with cationic organic probes to estimate the ΔΨ and thedegree of differential heating that will occur between normal andtransformed tissues. Dyes such as rhodamine 123, mitotracker green,calcein plus Co⁺⁺, 3,3′-dihexyloxacarbocyanine,triphenylmethylphosphonium,JC-1,5,5¹,6,6¹-tetrachloro-1,1¹,3,3¹-tetraethylbenzimidazolocarbocyanine,etc., all have an affinity for a negative mitochondrial ΔΨ. Based on theamount of cationic dye uptake, the membrane potential of specifictissue, tumors, and cells may be determined through the Nernst equation:ΔΨ=−(RT/F) ln(C_(in)/C_(out)). Which at physiologic conditions and 37°C. is =−61 log (C_(in)/C_(out)), where C_(in/out) is the concentrationof the probe inside or outside the mitochondria and plasma membrane. Byexample, a 10 to 1 gradient=−60 mV, 100 to 1=−120 mV. Uncouplersdissipate the ΔΨ, generate heat and release or prevent uptake ofcationic dyes. Six years of systematic measurement of mitochondrialmembrane potentials have been performed on human and mammalian cells,including some 200 cell types derived from human malignant tumors ofkidney, ovary, pancreas, lung, adrenal cortex, skin, breast, prostate,cervix, vulva, colon, liver, testis, esophagus, trachea and tongue.Based on this exhaustive study, a ΔΨ difference of at least 60 mV isknown to exist between normal epithelial cells and carcinoma cells. Thisis significant for the present invention in that uncoupling or “shortcircuiting” a 60 mV potential across a 5-nm mitochondrial membrane wouldbe equivalent to the amount of heat generated by short circuiting120,000 V across 1 centimeter. By exploiting or increasing the membranepotential between normal and transformed cells the rate of intracellularheat production by an uncoupler can be selectively increased in targettissues.

In order for uncoupler induced intracellular hyperthermia to be oftherapeutic benefit, the development of thermotolerance is also takeninto account in practicing this invention. Mammalian cells andprokaryotes acclimate and acquire transient resistance orthermotolerance to gradual or non-lethal hyperthermia. Such adaptationis believed to occur through increased synthesis of highly conservedgroups of proteins known as heat shock proteins (HSP). The amount of HSPpresent in tissues, cells and organisms subjected to non-lethal heat, orother forms of prolonged metabolic stress, is proportional to theirsurvival at higher temperatures. In general, thermotolerance developsafter 3 to 4 hours of continuous hyperthermia, peaks in 1 to 2 days anddecays back to normal thermosensitivy within 3 to 4 days.Thermotolerance is known to alter lethality of hyperthermia by as muchas 2° C. increase or double the heating time required to achieve thesame temperature-cytotoxic effect. Such adaptive thermoresistance byhuman tumors is problematic for continuous or fractionated cytotoxictreatment with hyperthermia. Induction heating times with the presentinvention are therefore kept to a minimum of 1 to 2 hours. Further, theuncoupler induced cytotoxic hyperthermia in the present inventioninduces relative tissue hypoxia, lowers intracellular pH and limits theproduction of ATP, all of which repress the development ofthermotolerance. Low doses of uncoupler, which produce gradual heatingcan be used to induce HSP synthesis and promote thermotolerance.

Determining the amount of DNP in mg/kg of body weight required toproduce the desired level of cytotoxic hyperthermia in a safe andefficacious manner is established from the thermal equivalents (Kcal) ofoxygen consumed (V0₂), and the known average specific heat capacity ofthe human body. It is known that at standard temperature and barometricpressure, 1 liter of oxygen consumed per minute (VO₂) generatesapproximately 4.862 Kcal. It is also known that the average specificheat capacity of humans is about 0.83 of that required to raise 1 gm ofH₂0 1° K=4.184 J, a heat capacity of 3.47 J g K⁻¹. An initial estimateof the total energy required to be generated by DNP to induce 41.0° C.hyperthermia in 1 hour may be very simply determined from the above andcustomized for a specific patient as outlined below:

Patient Characteristics

Body weight 70 kg Resting V0₂ 0.25 L/min Basal energy expenditure 73.1Kcal/hr (1754.4 Kcal/24 hrs.) Basal core temperature 37.0° C. Targettemperature 41.0° C.

Required energy to raise temperature to target level in 1 hour (Weightin grams=70×10³) (human specific heat=3.47 J g K⁻¹) (Temperatureincrease=41.0°-37.0° C.)˜0.97×10⁶ J. Since 1 J=4.184×10⁻⁴ Kcal, a totalpower input of about 232 Kcal would be required to raise the temperatureof the patient to the objective level in 1 hour less that amount of heatgenerated by a heated metabolism outlined below.

Increase in Metabolic Rate/Heat Production with Increase in BodyTemperature

The basal metabolic rate (BMR) is known to increase in patients withendogenous fevers by approximately 7% for each 0.5° C. rise intemperature. This is graphically depicted in FIG. 11 a. As a result, theincrease in BMR relative to the temperature will in itself assist inachieving the objective level during the induction phase by thefollowing equation:

BMR_(Tcore)=73.1×1.07^((Tcore-37)/0.5)

Thus, at 41.0° C. the metabolic rate will be 134.4 Kcal/hr, 61.3 Kcal/hrabove the basal energy expenditure level. This increase in metabolicrate will therefore reduce the initial energy required to heat thepatient by approximately 61 Kcal over the 1 hour timeframe.

Initial Net Energy Input Required to Reach Target Temperature in 1 Hour

232 Kcal−61 Kcal(by increased BMR)=171 Kcal

Required Increase in Initial V0₂ to Obtain 171 Kcal Heat Input

Since the Kcal equivalent for 1 liter of oxygen consumed per minute is4.862, then the initial increase in VO₂ required to generate 171 Kcalcan be calculated as follows: Heat in Kcal/min=V0₂×4.862. Since theindividual patient has a resting V0₂ of 0.25 l/min which=73.1 Kcal/hourBMR, then

X(V0₂)=171Kcal, or

X=0.25×171/73.1

An initial minimal increase in V0₂ to approximately 0.60 l/min isrequired.

DNP Dosage Required to Increase V0₂ to 0.60 l/min

The individual DNP dosage (mg/kg) required to produce an increase inoxygen consumption to 0.60 l/min so as to achieve a 171 Kcal heat outputis accomplished in the following fashion: (1) DNP is prepared in a 200mg/100 ml sterile aqueous solution. If not fully dissolved, it can bebrought into solution by buffering with 1% NaHCO₃, the pH must be keptbelow 8 to avoid hydrolysis; (2) the dose of DNP for each intravenousinfusion can vary from 0.5 to 4 mg/kg and will depend on the clinicalsituation, as well as the initial and subsequent increases in themetabolic rate (V0₂). In an especially preferred embodiment, the patientis given an initial dose of DNP no greater than 1 mg/kg intravenously,infused over no less than a 2 minute period. Within approximately 10-15minutes, a minimum of a 15% increase in V0₂ will occur. The V0₂ willcontinue to increase until a plateau is reached within an additional 5to 10 minutes. After a 5 minute plateau in V0₂, a subsequent dose ofeither 0.5, 1, 2, 2.5, or 3.0 mg/kg DNP is administered and V0₂ is againincreased until a desired plateau is reached. Additional infusions ofDNP or other medications are administered under clinical parameters ofV0₂, respiratory rate, pulse rate, blood pressure, urine output, cardiacoutput, core temperature, and clinical status of the patient so as tomaintain safe and effective control of heating. If heat dissipatingmechanisms are neutralized, measurable increases in core temperaturewill occur approximately 20 to 30 minutes after an increase in the V0₂.FIG. 13 illustrates the increases in V0₂ associated with repeatedinfusions of DNP.

Medications which increase the overall metabolic rate, or that ofspecific target tissues, and have short half-lifes can be utilized toincrease the relative activity of DNP or other uncouplers to furtheradjust V0₂ and heat production. Examples of such medications are almostlimitless because any drug, hormone or biologic response modifier thatcauses changes in enthalpy (heat content) during the course of itsintracellular chemical and biophysical activity and interaction in thelife cycle of biological cells can be utilized. A few illustrativeexamples include glucagon (half-life of 9 minutes in plasma), arbutamine(half-life 10 minutes), dobutamine (half-life 2 minutes), andvasopressin (half-life 5 minutes). Various amino acids and fatty acids,e.g., glutamine, proline, octanoate, etc., increase V0₂ by translocatingreducing equivalents into the mitochondrial matrix via themalate-aspartate shuttle, B-oxidation or proline metabolism. Agents suchas methylene blue (tetramethylthionine), ubiquinone, menadione,hematoporphyrin, phenazine methosulfate, 2,6-dichlorophenolindophenol,coenzyme Q1, CoQ2, or their analogs duroquinone and decylubiquinone,etc., can increase heat and/or free radical production by acting asartificial electron acceptors. Such agents, and numerous others, can beco-administered with DNP or other uncouplers to effectively increase theenthalpy changes in the entire organism or specific targeted tissues.

Minimizing Heat Loss and Temperature Control

Increased radiative and evaporative heat loss from man are the two mostdominant thermoregulatory mechanisms for cooling the body. The body'smethods of adjusting heat loss are vasoconstriction and vasodilation inthe skins blood vessels. Radiation can account for 60% of the heat lossgenerated by the body, while evaporation by sweating at 1.0 liter/hourcan represent a potential heat loss of about 1,000 Kcal/hour. By far,sweating and evaporation is the principal mechanism that dissipates heatunder conditions that induce large heat gains. Depending on the clinicalcircumstances, heat loss due to evaporation, as well as radiation, canbe managed and controlled by a variety of methods including, but notlimited to, using vasoconstricting agents, placing the patient in ascuba diving wet suit, humidified survival suit, or enveloping thepatient in a water soaked blanket covered or containing a polyethylenelining to prevent evaporative heat losses. Use of room ultrasonicnebulizers to induce continuous mist and high humidity is also known toprevent evaporative heat losses. Evaporative and radiant heat loss fromthe cranium is controlled by appropriate head gear, shower caps and/orwet towels. Control of local air velocities and management ofsurroundings as to temperature, emissivity, drafts, and convectioncurrents are important to avoid large heat losses. In those clinicalcircumstances where total body hyperthermia is required, failure toadequately control body heat loss will necessitate using higher doses ofDNP and induce a greater metabolic stress upon the patient.

If the core target temperature is exceeded or continues to rise afterthe target temperature is achieved, exposure of an extremity or bodysurface for a brief interval will permit sufficient heat loss to lowerthe core temperature to the target range. At target temperatures of39-41° C., residual uncoupling by DNP will continue for approximately 3hours. Heat production as a byproduct of glycolysis, and heatedmetabolism further maintains body heat content and compensates for anyheat loss. Therefore, target plateau temperatures can be regulated witha large margin of safety and with little to no additional use ofuncoupler. Therapy is terminated by removing the vapor barrier from thepatient. Evaporative and radiant heat loss from the patient generallyproduces a fall in core temperature of about 2-2.5° C. in about 20-30minutes. Obese patients and those with compromised thermoregulatorysystems experience a slower falloff in temperatures.

Patient Monitoring, Fluid Support and Evaluation During Treatment

Placement of physiologic monitoring sensors, intravenous fluids,supplemental oxygen (41/min) and optional oral diazepam sedation (5-10mg) is initiated prior to treatment. Patients receive 0.85 to 1.0 literof intravenous (IV) 5% dextrose in 0.25 normal saline per houralternated with 5% dextrose in 0.5 normal saline plus 7.5 to 10 meq ofKCl per liter to insure a urinary output of no less than 1 ml/kg/hr.Oxygen consumption, caloric expenditure, rectal core temperature,cardiac rhythm, blood pressure, heart rate and respiratory rate arecontinuously displayed, monitored by a trained member of the treatmentstaff. The data is automatically downloaded into a computer every 20seconds to 3 minutes for the entire procedure and immediatelyre-displayed on computerized graphs and charts. Two hours aftertreatment and 48 hours post-treatment, serum chemistries and hematologicprofiles are repeated. A typical patient flow chart is depicted in FIG.14.

Treatment of Excessive Heating and Antidotes

In those rare instances when too much uncoupler is administered or themetabolic rate of the patient unexpectedly increases and V0₂,hyperthermia, pulse rate and patient fatigue ensue, appropriatesupportive measures of cooling, intravenous hydration and administrationof specific medication should be instituted. Cooling should beinstituted by uncovering the patient, spraying with tepid water andfanning with an industrial grade fan. If cooling is inadequate, surface,axillary and groin ice packs and intravenous cold glucose solutionsshould immediately be considered. Bicarbonate, 1-2 mEq/kg should beadministered in the absence of blood gas analysis. Urine output of >1ml/kg/hour should always be maintained to avoid pre-renal azotemia andoliguria secondary to possible rhabdomyolysis and myoglobinuria.Mannitol should be administered if urine output is inadequate.Hypoglycemia should immediately be corrected with 50% saturatedintravenous glucose. If severe or persistent hypermetabolism ensues,rectal propylthiouracil-1,000 mg, hydrocortisone (100 mg q 6 h) ordexamethasone 2 mg q 6 h intravenously and/or sodium iodide as 1 gsodium ipodate (contrast agent) should be administered intravenously toinduce iatrogenic hypothyroidism. The decreased metabolic rate willdramatically reduce the physiologic response to DNP. Patient agitationand restlessness can be avoided by appropriate IV or IM dose ofdiazepam. Salicylates are of no value and may contribute to furtheruncoupling. Medications that reduce sweating, e.g., tricyclicantidepressants, antihistamines, anticholinergics, phenothiazines, ordecrease vasodilation, e.g., sympathomimetics, α-agonists, or decreasecardiac output, e.g., diuretics, beta-blockers or induce hypothalamicdepression, e.g., neuroleptics, α-blockers, opiods, etc., should beavoided prior, during and immediately after treatment with uncouplers.

The hypermetabolic and hyperthermic activity of DNP can furtherspecifically be reduced by using calcium channel blockers such asnifedipine, verapamil and others, in intravenous doses that do not causea drop in blood pressure or induce cardiac arrhythmias.Dihydrobenzperidol (a neuroleptic drug with a₁-adrenergic properties)can also be used to cause similar, significant reductions in DNP inducedhypermetabolism and hyperthermia. Dosages of these anti-DNP agents aretitrated in 5 mg to 30 mg increments and can be given either by mouth orintravenously. In those cases where DNP appears to decrease electricalconduction or cause EKG conduction abnormalities, Coenzyme Q10, in dosesof 50 mg/kg, can be used to restore normal electrical activity.

Patient Selection and Pretreatment Evaluation

It is imperative that in the practice of this invention, patients beselected and evaluated prior to treatment. Recommended patient inclusionand exclusion criteria includes: (1) patients have a definitivehistopathologic or other laboratory confirmed diagnosis of theirdisease; (2) the disease or condition should be responsive tointracellular hyperthermia treatment; (3) patients should have aKarnofsky score of 70% or greater; (4) not be pregnant; (5) weightshould be within 45% (+/−) of ideal body weight and patients must weighat least 35 kg; (6) there should be no history or findings ofanhidrosis, scleroderma, ectodermal dysplasia, Riley-Day Syndrome,arthrogryposis multiplex, extensive psoriasis, serious dysrhythmias,malignant hyperthermia or neuroleptic malignant syndrome,pheochromocytoma, hypocalcemia, repeated episodes of hypoglycemia,chronic or recurrent venous thrombosis, alcoholism, renal failure,cirrhosis, untreated hyperthyroidism, anaphylaxis associated with heator exercise-induced cholinergic type urticaria, exercise or heat inducedangioedema, schizophrenia, catatonia, seizure disorders, emotionalinstability, Parkinson's disease, brain irradiation, cystic fibrosis,unstable angina pectoris, congestive heart failure, patients withcardiac pacemakers, severe cerebrovascular disease, spinal cord injury,severe pulmonary impairment, hereditary muscle disease such as Duchennetype muscular disease, central core disease of muscle, myotoniacongenita, King-Denborough syndrome, Scwanry-Jampol syndrome, orosteogenesis imperfecta; (6) no immediate use of drugs that impair thebody's heat dissipation mechanisms such as phenothiazines,anticholinergics, antihistamines, antiparkinsonians, glutethimide,hallucinogens, lithium, cocaine or other illicit drug use, monamineoxidase inhibitors, sympathomimetics, phencyclidine, opioids,phenylephrine, INH, tricyclic antidepressants, withdrawal from dopamineagonists, or cardiovascular drugs that clinically impair cardiac outputor thermoregulatory vasodilation such as high doses of β-blockers,vasodilators, or calcium channel blockers; and, (7) the patient shouldnot be anemic or otherwise have a reduced oxygen absorbing, carrying orutilizing capacity.

Pretreatment evaluation should include a complete medical history andphysical examination focused on the selection criteria listed above.Laboratory evaluation should include pulmonary function tests-ifindicated, full hematological survey with hemastatic profile, EKG, liverfunction tests, serum biochemical profile, thyroid panel, serumcreatinine, calcium, phosphate, and stress-EKG or exercise-multigatedradionucleotide ejection scan on patients whose cardiac ejectionfraction is suspect not to be greater than 45% with probabledeterioration on exercise. While clinical exceptions to entry laboratoryvalues may exist, the following laboratory data should be a benchmarkguide for initiation of treatment: hemoglobin >=11.0 g/dl for menand >=10.0 g/dl for women, platelet count >=75,00 platelets/mm³,bilirubin <=2×ULN (ULN=upper limit of normal), ALT (SGPT)<=2×ULN, AST(SGOT)<=2×ULN, pancreatic amylase <1.5×ULN, neutrophil count >=1,000cells/mm³. Serum electrolytes and K⁺ should be well within normallimits, as hypokalemia decreases muscle blood flow, cardiovascularperformance, and sweat gland function.

More generally, the method outlined above is to be tailored to anindividual patient. As set forth above, the DNP may be administered byintravenous infusion. Alternatively, the route of administration mayalso be orally, rectally or topically. The frequency and optimal timeinterval between administrations is individualized and determined bymeasuring V0₂, as well as other parameters. For example, variouslaboratory, x-ray, CAT scan, MRI, PET scan, HIV load, CD4+ lymphocytecounts, HSP expression, prostatic specific antigen (PSA) and othersurrogate markers of clinical outcome can establish the V0₂, frequencyand duration of therapy. One treatment, or treatments as frequent asevery day, or every other day, as far apart as 1 year or longer may berequired for sustained beneficial results.

The optimal V0₂, temperature, duration, and frequency between treatmentswill probably vary from patient to patient and the specific disease orcondition being treated. One skilled in the art would be able to modifya protocol within the present invention, in accordance with standardclinical practice, to obtain optimal results. For example, the HIVrelationships between viral load, CD4⁺ lymphocyte counts, presence ofopportunistic infections and clinical status of the patient can be usedto develop more optimal regimes of DNP administration. Applicants'studies have revealed that the methods of the present invention can beeffective in the diagnosis and treatment of a wide range of diseasestates and conditions in which uncoupler induced hypermetabolism,hyperthermia, oxidative stress and their sequela, play a beneficialrole. To those skilled in the art, it is also encompassed that a varietyof different veterinary, as well as medical, applications for treatmentand diagnosis can be practiced with the present invention.

It is envisioned that DNP, or other uncouplers, may also be administeredwith other compounds used to treat infectious, malignant or otherdiseases. Examples of other agents include antifungal, antibacterial,antiviral or anti-neoplastic drugs, cell differentiating agents, and,various biologic response modifiers. Examples of anti-fungal agentsinclude Amphotericin B, Griseofulvin, Fluconazole (Diflucan),Intraconazole, 5 fluoro-cytosine (Flutocytosine, 5-FC), Ketatoconazoleand Miconazole. Examples of anti-bacterial agents include antibiotics,such as those represented from the following classifications: betalactam rings (penicillins), macrocyclic lactone rings (macrolides),polycyclic derivatives of napthacenecarboxamide (tetracyclines), aminosugars in glycosidic linkages (aminoglycosides), peptides (bacitracin,gramicedin, polymixins, etc.), nitrobenzene derivatives ofdichloroacedic acid, large ring compounds with conjugated double bondsystems (polyenes), various sulfa drugs including those derived fromsulfanilamide (sulfonamides, 5-nitro-2-furianyl compounds (nitrofurans),quinolone carboxylic acids (nalidixic acid), fluorinated quinilones(ciprofloxan, enoxacin, ofloxacin, etc.), nitroimidazoles(metroindazole) and numerous others. These antibiotic groups areexamples of preferred antibiotics, and examples within such groupsinclude: peptide antibiotics, such as bacitracin, bleomycin,cactinomycin, capreomycin, colistin, dactinomycin, gramacidin A,enduracitin, amphomycin, gramicidin J, mikamycins, polymyxins,stendomycin, actinomycin; aminoglycosides represented by streptomycin,neomycin, paromycin, gentamycin ribostamycin, tobramycin, amikacin;lividomycin beta lactams represented by benzylpenicillin, methicillin,oxacillin, hetacillin, piperacillin, amoxicillin and carbenacillin;lincosaminides represented by clindamycin, lincomycin, celesticetin,desalicetin; chloramphenicol; macrolides represented by erythromycins,lankamycin, leucomycin, picromycin; nucleosides such as 5-azacytidine,puromycin, septacidin and amicetin; phenazines represented by myxin,lomofungin, iodin; oligosaccharides represented by curamycin andeverninomycin; sulfonamides represented by sulfathiazole, sulfadiazine,sulfanilimide, sulfapyrazine; polyenes represented by amphotericins,candicidin and nystatin; polyethers; tetracyclines represented bydoxycyclines, minocyclines, methacylcines, chlortetracyclines,oxytetracylcines, demeclocylcines; nitrofurans represented bynitrofurazone, furazolidone, nitrofurantoin, furium, nitrovin andnifuroxime; quinolone carboxylic acids represented by nalidixic acid,piromidic acid, pipemidic acid and oxolinic acid. The Encyclopedia ofChemical Technology, 3rd Edition, Kirk-Othmer, editors, Volume 2 (1978),which is hereby incorporated by reference in its entirety.

Antiviral agents that can be used with DNP include: interferons α, β andγ, amantadine, rimantadine, arildone, ribaviran, acyclovir, abacavir,vidarabine (ARA-A) 9-1,3-dihydroxy-2-propoxy methylguanine (DHPG),ganciclovir, enviroxime, foscarnet, ampligen, podophyllotoxin,2,3-dideoxytidine (ddC), iododeoxyuridine (IDU), trifluorothymidine(TFT), dideoxyinosine (ddi), d4T, 3TC, zidovudine, efavirenz, proteaseinhibitors such as indinavir, saquinavir, ritonavir, nelfinavir,amprenavir, etc., and specific antiviral antibodies.

Anti-cancer drugs that can be used with DNP include, but are not limitedto, various cell cycle-specific agents represented by structural analogsor antimetabolites of metholtrexate, mercaptopuorine, fluorouracil,cytarabine, thioguanine, azacitidine; bleomycin peptide antibiotics,such as podophyllin alkaloids including etoposide (VP-16) and teniposide(VM-26); and various plant alkaloids such as vincristine, vinblastine,and paclitaxel. Anti-neoplastic cell cycle-nonspecific agents such asvarious alkylating compounds such as busulfan, cyclophosphamide,mechlorethamine, melphalan, altaretamine, ifosfamide, cisplatin,dacarbazine, procarbazine, lomustine, carmustine, lomustine, semustine,chlorambucil, thiotepa and carboplatin. Anticancer antibiotics andvarious natural products and miscellaneous agents that can be used withDNP include: dactinomycin, daunorubicin, doxorubicin, plicamycin,mitomycin, idarubicin, amsacrine, asparaginase, quinacrine, retinoicacid derivatives (etretinate), phenylacetate, suramin, taxotere,tenizolamide, gencytabine, amonafide, streptozocin, mitoxanthrone,mitotane, fludarabine, cytarabine, cladribine, paclitaxel (taxol),tamoxifen, and hydroxyurea, etc.

DNP can also be administered with various hormones, hormone agonists andbiologic response modifying agents which include, but are not limitedto: flutamide, prednisone, ethinyl estradiol, diethylstilbestrol,hydroxyprogesterone caproate, medroxyprogesterone, megestrolacetate,testosterone, fluoxymesterone and thyroid hormones such as di-,tri- andtetraiodothyroidine. The aromatase inhibitor, amino glutethimide, thepeptide hormone inhibitor octreotide and gonadotropin-releasing hormoneagonists such as goserilin acetate and leuprolide can also be used withDNP. Biologic response modifiers such as various cytokines, interferonalpha-2a, interferon alpha-2b, interferon-gamma, interferon-beta,interleukin-1, interleukin-2, interleukin-4, interleukin-10, monoclonalantibodies (anti-HER-2/neu humanized antibody), tumor necrosis factor,granulocyte-macrophage colony-stimulating factor,macrophage-colony-stimulating factor, various prostaglandins,phenylacetates, retinoic acids, leukotrines, thromboxanes and otherfatty acid derivatives can also be used with DNP.

The use of this invention should be under the strict direction of aqualified and specialized treatment team to insure safety andeffectiveness. The treatment team remains with the patient throughoutthe procedure to insure that safe and controlled dosages of an uncouplerare administered by monitoring real time changes in V0₂, metabolic rate,temperature, respiratory rate, heart rate, urine output and clinicalstatus of the patient. This invention is practiced in controlled stepsso as to attain a predetermined V0₂ and plateau of heating time for aparticular disease or condition. For example, in cases were heatdissipation mechanisms do not have to be blocked, the specialized teamwill periodically recheck V0₂, heart rate, blood pressure, CAT scan,MRI, etc., and other laboratory and clinical parameters to insurecontinued safety and efficacy of DNP therapy. It is preferred that thespecialized team undergo a training period in the use of this inventionprior its administration to human patients.

The present invention is further illustrated by reference to thefollowing examples, which illustrate specific elements of the inventionbut should not be construed as limiting the scope of the invention.

Example 1 Method of Using DNP with Glucagon to Treat ParasiticInfections, Hydatid Disease of the Liver

History: A 52 year old white Swiss male, European fox hunting dogtrainer, presented with right upper quadrant pain and vomiting. Pasthistory revealed he had hepatic “cyst” surgery 2 years ago.Preoperatively, he was treated with albendazole. Only one dose ofalbendozale was given because of a “near death” anaphylactic reaction.He denied history of weight loss, pulmonary, cardiac, neurologic orthermoregulatory problems. There was no history of alcohol abuse ormedication use. The patient was adamantly opposed to any further surgeryor treatment with albendazole or mebendazole.

Physical Examination: Weight=90 Kg; height=177.8 cm; BP=140/80; HR=76 &reg; Resp.=18 min; T=37.0

An old well healed scar consistent with prior hepatic surgery waspresent. Physical exam otherwise was unremarkable.

Laboratory studies: EKG, chest X-ray, blood panel, including serumelectrolytes, thyroid studies and liver function tests were withinnormal limits (WNL). A complete blood count was unremarkable except for20% eosinophilia. Ultrasound and nuclear magnetic resonance revealed 4,2 to 3 cm. in diameter, cysts in the right middle lobe of the liver anda solitary 2 cm semi-solid medullary cyst in the neck of the righthumerus. ELISA serology showed a diagnostic titer for hydatid disease.Review of previous surgical liver pathology reports revealed a cestodecompatible with Echinococcus multilocularis.

Clinical assessment and treatment evaluation: The patient had nohistorical or physical contraindications to DNP induced hyperthermia.Conventional therapy of hydatid disease is either surgical resection ormedical therapy with albendazole for 4 weeks. Hydatid bone cysts are notamenable to surgery and respond poorly to standard medical therapy.Echinococcus multilocularis protoscoleces and the germinal membranes ofhydatid cysts are known to be irreversibly destroyed by heating at 41°C. for 15 minutes. Human liver and hepatocytes can withstand artificialtemperatures of 42° C. for as long as 20 hours without irreversibledamage. Acute glucagon treatment is known to preferentially stimulatehepatocyte mitochondrial V0₂. Rates of hepatocyte uncoupled V0₂ are alsoknow to be stimulated up to 100% in less than 6 minutes after thehormonal action of glucagon. Acute glucagon treatment has been shown toselectively increase the pH gradient across hepatocyte mitochondrialmembranes. Thus, it can be empirically presumed that any increase in V0₂from glucagon administration causes increased thermogenesis,predominantly in the liver.

Pretreatment protocol: the patient was given 10 mg diazepam by mouth anddressed into a modified wet suit. The wet suit was cut lengthwise at thearms and legs. Velcro strappings were attached at the cuttings forclosure, rapid removal or exposure of the limb(s). After placement ofmonitoring sensors, he was started on IV fluids of 5% dextrose, 0.5normal saline with 7 meq K⁺, infused at an initial rate of 12 cc/kg/hr.Evaporative heat loss from the head was minimized by a plastic showercap and towels. A 401AC temperature probe (YSI Incorporated, YellowSprings, Ohio) was inserted 11 cm. into the rectum. The probe wasconnected to a Model 4600 telethermometer (YSI 4600 PrecisionThermometer) and readings within 0.1° C. were continuously displayed andrecorded at baseline and during treatment on Hewlett-Packard (HP)computer systems with customized software developed by MR&S (Manalapan,N.J.). A TEEM 100 Metabolic Analysis System (AeroSport Inc., Ann Arbor,Mich.), with a modified face mask and oxygen delivery system (38-40% O₂saturation) for patient comfort and increased accuracy, was attached tothe patient. Oxygen consumption (V0₂), carbon dioxide production (VCO₂),expired air volume (V_(E)), heart rate (HR), and Kcal of heat producedwere measured in 20 second intervals and extrapolated to minute orhourly rates. All patient data was monitored in real time, continuouslydisplayed at baseline and during treatment and recorded on HP computersystems with customized software from MR&S (Manalapan, N.J.).

Treatment procedure: After baseline recordings of 10 minutes, therequired amount of DNP to raise the initial V0₂ to achieve a temperaturein the patient of 40° C. was calculated as described under “DNP dosagerequired to increase V0₂”. The patient was given an initial dose of 1mg/kg of DNP, infused intravenously over a 3 minute period. After theV0₂ stabilized at 40% above baseline, an additional DNP infusion of 3mg/kg was given. Upon attaining a stable V0₂, 0.5 mg of glucagon wasadministered intravenously. After this stabilization of V0₂, a glucagondrip was variably infused from 0.5 to 5 mg/kg/hour to additionallycontrol V0₂ and selectively augment heat production in the liver. Thetreatment procedure was discontinued after the patient was maintained ata rectal body temperature of 40° C. for about 1 hour. The wet suit wasopened and head covering removed. After the patient's body temperaturereached 38° C., the Foley catheter was removed and intravenous fluidswere discontinued. Evaporative and radiant heat loss lowered the bodytemperature to a normothermic level within 30 minutes. No immediate ordelayed post-treatment toxicity was encountered. Monitored patientparameters are shown in FIG. 15.

Treatment outcome: Serial imaging studies revealed hepatic and bone cystshrinkage with increased density at 2 and 4 weeks post treatment. Repeatmagnetic resonance imaging at 4 months showed complete cystdisappearance in the liver and bone.

Example 2 Method of Using DNP to Treat Viral Infections, HIV Disease

History: A 38 year old white male, past intravenous heroin addict, wasdiagnosed approximately 8 years ago with HIV by ELISA and positiveWestern blot for HIV p24 and gp41 antigens after presenting with weightloss and thrush. His history included repeated treatment forcandidiasis, pneumocystis carinii, and various subcutaneous abscesses.Past medications included sulfamethoxazole, ketoconazole, fluconazole,zidovudine, didanosine and various other antibiotics. For the past yearand a half he has been on highly active antiretroviral therapy (HAART)with various HIV protease inhibitors combined with thymidine, purine orcytosine nucleoside and nonnucleoside inhibitors. He was unable totolerate nelfinavir because of diarrhea. Ritonavir caused intractablevomiting and abdominal pain. Current medications include indinavir,zidovudine and lamivudine. Review of the most recent viral load (VL) andCD4+ lymphocyte counts showed an initial drop in plasma HIV RNA(copies/ml) from 200,000 to 2,000 over a 12 week period with the VLrebounding back to 200,000 at week 16. CD4+ lymphocyte counts haveremained between 100 to 200 cells/mm³. Approximately 5 months ago he wastreated for oral and endobronchial Kaposi's sarcoma (KS) with liposomaldaunorubicin followed by liposomal doxorubicin. He denied treatment withvincristine or bleomycin. There is no history of recent diarrhea, recentweight loss, hemoptysis, shortness of breath on moderate exertion, orcardiac problems. There has been no illicit drug use over the past 2years. The patient stated no combination of HAART has been able to lowerhis viral load and multiple side effects from the drugs are limiting hiscompliance to take the medications. There was no history ofthermoregulatory problems.

Physical examination: weight=60 Kg; height=155 cm; BP=128/72; Resp=20;T=38.2° C.; and, the pulse was 92 & reg. Exam revealed asthenia andgeneralized enlargement of lymph nodes, some 2 to 3 cm in diameter inthe axillary and inguinal regions. There was diffuse oropharyngealthrush. Beneath the thrush, the oral cavity also contained several darkred plaque to nodular like lesions on the hard palate and gingiva. Thelesions did not blanch on compression with the tongue blade. A crustedstrawberry like mass, 1 by 2 cm, was present at the anus. There were noneurologic deficits or ocular lesions.

Laboratory studies: EKG, serum electrolytes, renal and liver functiontests were normal. Hematocrit was 35.5%, WBC was 9,900 with 81%neutrophils, 4 bands, 11 lymphocytes and 4 monocytes. Platelets were314,000/mm³. Viral load was 400,000 copies/ml (Amplicor HIV Monitortest, Roche). A CD4⁺ T cell count was quantified by flow cytometry at250/mm³. He was antibody positive for hepatitis C. Chest radiographshowed some bilateral apical patchy opacities. Pulmonary function testsshowed all parameters, including forced expired volume, greater than 80%of predicted. Karnofsky score was greater than 70. Normal and tumortissue biopsies, 3 to 6 mm in diameter, from the oral cavity and anuswere obtained. The tissues were equally divided, weighed and placed in4° C. Ringers lactate solution. Histologically confirmed normal and KStissues were then subjected to microcalorimetric measurements in athermal activity monitor (ThermoMetric, Jarfalla, Sweden). Recorded heatoutput (μW/min) was 8.2-8.5 times greater for the KS sarcoma lesionsthan nontumorous oral mucosa. tissues. Repeat measurements with biopsiesspecimens in 30 uM DNP increased heat production in tumorous tissues20.5 times more than nontumorous specimens.

Clinical assessment and treatment evaluation: HIV and HIV-infected Tcells are known to be more sensitive to killing by heat than uninfectedlymphocytes. Susceptibility to heat killing is enhanced with increasedoxygen free radical production. Acute and chronically infected cellshave decreased levels of manganous superoxide dismutase (MnSOD)activity. MnSOD is located exclusively in mitochondria. Mathematicalmodeling of human HIV production and CD4+ T cell turnover predicts thatreducing both free virus and actively infected cells by a minimum of 40%with 1 hour of 42° C. therapeutic hyperthermia every third day willpromote recovery of the uninfected T-cell population. Human HIV studieswith extracorporeal hyperthermia of 41-42° C. have reported isolatedcases of extended patient survival, elimination of detectable virus, andimprovement of Kaposi's sarcoma lesions. DNP is known to generateintracellular hyperthermia and oxygen free radicals from the level ofthe inner mitochondrial membrane. Studies on in vitro inactivation ofchronically HIV infected HUT-78 cells by various concentrations of DNPare graphically represented in FIG. 16.

The patient has been and remains resistant to treatment with HAART.Opportunistic infections with candida and Kaposi's sarcoma herpes virus(KSHV, human herpesvirus type 8) causing his thrush and Kaposi's sarcomaare comorbid conditions indicative of a worsening prognosis. In spite ofhaving AIDS with candidiasis and Kaposi's sarcoma, the patient maintainsgood cardiac and pulmonary function. There was no history ofthermoregulatory problems. It was discussed and agreed that hyperthermiatreatments with core body temperatures of 41° C. would be administeredon a daily or every other day basis, as tolerated, for a minimum of 3hours, not to exceed 5 hours.

Pretreatment protocol: all medications were stopped 2 weeks prior totreatment. The patient refused taking diazepam, placement of a Foleycatheter and oxygen face mask. He dressed himself into a dry cold waterimmersion suit (Stearns, ISS-590I, Universal Adult) designed to preventheat loss and modified for easy placement of physiologic monitors.Equipment for measurement of heart rate, temperature, carbon dioxideproduction and Kcal of heat produced were conducted as outlined inExample 1. An oral breathing tube was used to measure V0₂ from room air.Urine output was measured when the patient voluntarily urinated througha “Texas” catheter (superficial condom tightly fitted around the head ofthe penis with tubing connected to urine collection bag). The patientwas informed that hyperthermia would be administered as tolerated by hisstamina and monitored clinical parameters, not to exceed 5 hours, on adaily or every other day basis, for a total of 5 sessions.

Treatment procedure: Baseline reading for 5 minutes established anaverage V0₂ of 300 cc/min. An initial dose of 2 mg/kg of DNP wasadministered over a 2 minute period. V0₂ increased and stabilized at 15minutes at 340-380 cc/min. An additional 2 mg/kg DNP infusion was given,the V0₂ increased and stabilized at 610-630 cc/min. Body coretemperature increased to 39.4° C. within 60 minutes. A gradual fall inblood pressure was noted at 90 minutes to 100/60 mm/Hg. Norepinephrinebitartrate (Levophed) was given IV drip at a dose of 1 microgram/min.and adjusted to maintain blood pressure at 130/80. Approximately 1minute after initiating the vasopressor, heart rate increased from 90 to100 and V0₂ to 0.85 liters/min. Core body temperature increased within20 minutes to 41.5° C. V0₂ was maintained at 1.0 liters/min. by loweringor increasing the dose of norepinephrine. An additional infusion of 1mg/kg DNP was given at hour 4 to correct a dropping V0₂. On occasionswhen the core temperature increased above 41.6° C., a lower extremitywas exposed for evaporative heat loss. The patient withstood theprocedure without any untoward effects for a period of 7 hours. Theprotocol was repeated consecutively for 5 days without the additionaluse of vasopressors.

Treatment outcome: Immediately after the first treatment oralcandidiasis improved by 50%. The oral and anal Kaposi's lesionsexhibited marked erythema with circumferential areas of blanching. Onthe second day of treatment the KS erythema diminished. There was noevidence of oral candidiasis on the 3^(rd) day of therapy. The analtumor was crusted and approximately 60% diminished in size on the 5^(th)and last day of therapy. Lymphadenopathy progressively decreased and wasresolved at 2 weeks post-treatment. At 30 days post-treatment, there wascomplete regression of both oral and anal KS lesions. Repeat blood workon days of treatment showed no significant hematologic, electrolyte,liver or kidney changes from baseline. Viral load immediately aftertreatment day 5 showed 50,000 HIV-RNA copies/ml. HIV RNA wasnon-detectable at 4, 6 and 12 weeks post-treatment. CD4+ T celllymphocyte counts increased to 380-420 cells/mm³ by week 4 and remainedstable at week 6 and 12. FIG. 17 shows monitored patient parameters ontreatment day 1. FIG. 17 a) shows changes in surrogate markersimmediately after treatment, weeks 4, 6 and 12.

Example 3 Use of DNP to Treat Bacterial Infections, Lyme Disease

History: A 33 year old white female with a textbook case of Lymeborreliosis related being bitten by a tick and developing apathognomonic erythema migrans on her right anterior thigh. The rashresolved within two weeks but 3 months later she developed verbal memoryimpairment, migratory arthritis of the knees, ankles and tibias.Fibromyalgias, tachycardias and a left sided Bell's palsy ensued.Constitutional symptoms of fatigue, malaise and severe depression causedher to undergo psychiatric care for 1½ years before she was definitivelydiagnosed with chronic Borrelia burgdorferi infection. She was treatedwith ceftriaxone, 2 g intravenously every 12 hours for 14 days. Fourmonths after apparent improvement she developed photophobia, headaches,pronounced memory loss, depression, dysesthesias and a painful, swollenleft knee joint. Repeat ELISA, Western blot and DNA-PCR were allpositive for B. burgdorferi. Spinal tap showed pleocytosis with positiveantibody and PCR tests for neuroborreliosis. Over the next year thepatient received prolonged ceftriaxone, 2 g per day intravenously for 3months, and 3 individual short courses of oral ciprofloxacin,minocycline, and azithromycin. Symptoms failed to resolve. Two monthsafter her last regimen of antibiotics a new annular erythematouseruption, suggestive of erythema migrans, reoccurred on the right thighand developed under her left axilla. Doxycycline was instituted and therash subsided. The patient refused further antibiotic therapy because ofassociated intractable diarrhea and has made tentative plans to undergo“malariotherapy” in China.

Physical examination: weight=60 Kg; height=160 cm; BP=130/70; HR=86 &reg; resp=18; T=37.3° C. Physical exam revealed a swollen and tenderleft knee. A thin, atrophic hypopigmented area of skin over the rightthigh, typical of acrodermatitis chronica atrophicans was present.Neurologic exam showed some verbal memory deficit. There were bilateral,lower distal extremity paresthesias.

Laboratory studies: EKG demonstrated a first-degree atrioventricularblock (PR internal >0.2 sec), some widening of the QRS complex andWenckebach periodicity. There were no dropped beats. Left kneearthroscopy showed synovial hyperthrophy with early erosive arthritis.Synovial fluid analysis revealed a WBC of 50,000 cells/ml with 70%neutrophils and a positive DNA-PCR for Borrelia burgdorferi. Biopsysections of synovial tissue showed chronic nonspecific synovitis.Warthin-Starry and silver staining histology revealed spirochetalorganisms consistent with Borrelia burgdorferi. Lumbar puncture spinalfluid analysis showed pleocytosis, elevated gamma globulin and positivePCR for B. burgdorferi. Spinal fluid cultured for 2 months inBarbour-Stoenner-Kelly medium was reported positive for B. burgdorferi.Serum electrolytes, kidney, liver function and hematologic studies wereall within normal limits. The patient underwent a stress EKG, attaininga maximum heart rate of 165 with no evidence of arrhythmia or S-Tsegment depression.

Clinical assessment and treatment evaluation: Lyme disease is a zoonosiscaused by a slow growing pathogenic spirochete, Borrelia burgdorferi. Invarious mammalian species, including man, these organisms are known toinvade heart, kidneys bladder, spleen and brain. Borrelia spirochetesare very resistant to treatment with antibiotics, especially if there isevidence of central nervous system or joint involvement. Viable B.burgdoferi have been isolated from antibiotic treated monolayers offibroblasts. Borrelia spirochetes are known to be facultativeintracellular pathogens in fibroblasts by laser scanning confocalmicroscopy. Central nervous system tissue, joints, front chamber of theeye and intracellular location can provide the Lyme spirochete with aprotective environment against antibiotic therapy and Borreliaburgdorferi have been reliably cultured from patients with chronicdisease, even from those previously aggressively treated. This patienthas confirmed chronic CNS and joint Lyme disease in spite of extensiveantibiotic therapy.

The Lyme spirochete is irreversibly inactivated by heating at 40° C. for3 hours, 41° C. for 2 hours or 41.5° C. for 1 hour. Susceptibility ofall strains of Borrelia burgdorferi to penicillin and ceftriaxone isincreased up to 16-fold by elevation of temperature from 36° C. to 38°C. At 40° C. Borrelia burgdorferi increases expression of at least 12heat shock proteins (HSP), most of which are strongly immunogenic. Thepatient had no history of thermoregulatory problems. She was informedthat her body temperature would be raised between 40 to 41° C. for aperiod of 3 hours, the actual level and time under hyperthermia woulddepend on her monitored clinical parameters.

Pretreatment protocol: the evening prior treatment the patient wasinstructed not to eat and dress in cotton undergarments. Approximately 4hours prior to treatment 2 mg alprazolam was administered by mouth. Thepatient dressed herself into a dry cold water immersion suit (Stearns,previously described) with headgear. Monitoring sensors, including EKGdisplay, IV fluids and Foley catheter were attached and the suit waszipped closed. The patient opted for oxygen supplementation. Themodified face mask was connected to the TEEM 100 metabolic AnalysisSystem for V0₂ measurements. Data was recorded as previously described.

Treatment procedure: baseline recordings of 10 minutes showed a V0₂ of220 cc/min., 3.7 cc O₂/kg/min. The patient was infused with 1 mg/kg DNPover a 2 minute period. V0₂ increased and stabilized at 250 cc/min, 5.3cc/kg/min. A second dose of 2.0 mg/kg was infused over a 2 minute periodand the V0₂ peaked at 400 cc/min, 8.8 cc O₂/kg/min. An additional doseof 1.0 mg/kg DNP was given 30 minutes after the second dose. The V0₂increased and reached a stable plateau at 600 cc/min, 10.8 cc/kg/min.Rectal temperature continued to climb until a range of 40.2 to 40.6° C.was reached at 70 minutes after the initial dose. A fall in V0₂ wasnoted at 90 minutes, a dopamine drip at 2-3 mcg/kg/min was initiated.V0₂ increased back to 680-710 cc/min. The temperature remained stablebetween 40.1° C. and 40.6° C. throughout the 3 hour plateau treatmentperiod. The patient periodically requested the V0₂ monitoring mask beremoved during the hyperthermia treatment period. She was accommodatedwith removal of the mask on two occasions for periods not exceeding 10minutes. The patient experienced no problems during the procedure butwas noticeably fatigued by hour 3. The treatment was terminated 4 hoursand 10 minutes after the initial dose of DNP. Twenty five minutes afterthe patient was removed from the neoprene survival suit, the rectal coretemperature dropped to 38.5° C. Normothermia was achieved approximately60 minutes after cessation of therapy and removal from the survivalsuit. Approximately 6.5 to 7 hours after treatment the patientexperienced chills, an increase in oral temperature to 38.7 degreescentigrade and malaise. IV fluids and the dopamine drip at 2 mcg/kg/minwere restarted and the patient was closely observed. Her symptomssubsided over 3 hours and by the next day she felt active and hungry. Itwas surmised she may have experienced a delayed Jarisch-Herxheimerreaction. The patients monitored treatment flow chart is FIG. 18.

Treatment outcome: at two months follow-up the patient stated herarthralgias, myalgias, malaise, fatigue and memory deficits havedisappeared. Lower extremity dysesthesias were no longer present. EKGshowed resolution of her first degree A-V block. The patient wasinformed of her past positive cerebrospinal fluid positive culture forthe Lyme disease spirochete. It was suggested a repeat spinal tap beperformed for B. burgdorferi by PCR and culture. If positive, thepatient agreed she would be re-treated with both DNP inducedhyperthermia and intravenous ceftriaxone for maximum synergism. Repeatspinal fluid analysis was normal, i.e., no elevated protein, nodetectable Borrelia DNA by PCR and no pleocytosis. Three months later,spinal fluid culture on Barbour-Stoenner-Kelly II medium was reportednegative.

Example 4 Method of Using DNP with Vasopressors and Chemotherapy toTreat Neoplasia, Peritoneal Carclnomatosis

History: A 55 year old female presented with a distended abdomen due toascites. Laparotomy revealed peritoneal dissemination of a malignancywith histological findings of an undifferentiated adenocarcinoma, originunknown.

Physical examination: weight=55 kg; height=154 cm; BP=140/90; HR=88 &reg; Resp=22; T=37.6° C. The patient was a well developed and wellnourished Muslim female with a healing midline laparotomy scar.Ballotable ascites was detected in the abdomen. There was nolymphadenopathy.

Laboratory studies: laboratory examination of the ascitic fluid showedhigh levels of amylase. She had a hemoglobin of 9.2. High levels ofamylase and tumor markers, including CA15-3, CA 125 and CA72-4 werepresent in the serum. Blood chemistry, liver and kidney function testswere within normal limits. Chest X-ray and EKG was normal. MRI andultrasound of the abdomen showed normal pancreas, liver and atrophicovaries, there were widespread nodular lesions consistent withperitoneal carcinomatosis.

Clinical assessment and treatment evaluation: the patient had aninoperable malignancy of unknown origin. Chemotherapy in such cases isonly of marginal survival benefit. Hyperthermia, combined withchemotherapy has been shown to be synergistic with increased tumorresponse and survival benefit. Tumor antigen markers are known to beincreased by the heat shock response and may further enhance immunologicsurveillance. The patient had no history of thermoregulatory problemsbut refused to be placed in wet suit or survival suit because of a“phobia of enclosed tight garments”.

It was elected to treat the patient with hyperthermochemotherapy.Treatment consisted of DNP, and combination chemotherapy withcarboplatin, mitomycin, and doxifluridine. An α-1 adrenergic receptoragonist was used to minimize peripheral vascular dilation and heat loss.

Pretreatment protocol: the patient was transfused with three units ofpacked red blood cells. A Foley catheter was inserted on each day oftreatment. She was covered in a water soaked blanket containing apolyethylene lining A shower cap with towels was used to prevent heatloss from the head. Intravenous lines were placed into both arms with 19gauge intracaths. EKG, heart rate, rectal thermistor, and V0₂ monitorswere attached. Oxygen supplemented facemask and equipment was attachedand data monitored as previously described under Example 1.

Treatment protocol: the patient was given chemotherapy by mouth. Thetotal doses of carboplatin, and mitomycin were 450 mg and 24 mg IVrespectively on day 1 and last day of week 6. Doxifluridine, 600 mg, wasorally administered every day for 5 days and repeated the last 5 days ofweek 6. On the day of DNP infusion, baseline recordings were establishedfor 10 minutes. Mephenteramine sulfate, 30 mg, was given byintramuscular injection. Ten minutes later her heart rate increased to96 and her V0₂ increased from 250 to 320 cc/min. V0₂, heart rate andblood pressure stabilized after 20 minutes and she was given an initialdose of 1 mg/kg DNP. Additional 0.5 mg/kg infusions of DNP wereadministered in 3 successive infusions spaced 20 minutes apart. Thepatients V0₂ stabilized between 780-820 cc/min. and her core temperatureincreased to a maximum of 41.4° C. After a plateau temperature of 41.5°C.±0.5° C. was reached, her level of V0₂ and temperature was maintainedfor a period of 2 hours and 30 minutes with an additional infusion of0.5 mg/kg DNP given 50 minutes after the last dose. The DNP treatmentprotocol was repeated every fourth day for a period of 6 weeks. Arepresentative monitored flow chart is shown in FIG. 19.

Treatment outcome: By the combined treatments outlined above, ascitesresolved by the end of the sixth week. Serum levels of amylase and alltumor markers decreased after the third week of treatment and werenormal at week 6. Repeat magnetic resonance imaging and echore-examination of the abdomen showed complete resolution of peritonealmetastasis. Nine and a half months after treatment, the patient is alivewithout any evidence of tumor reoccurrence.

Example 5 Use of DNP with Thermosensitive Liposomes

To overcome the toxicity to normal tissues of many anticancer agentssuch as doxorubicin and anti-infectious drugs such as amphotericin B,liposomal formulations have been developed. Liposomal doxorubicin isknown to have reduced cardiotoxicity and increased antineoplasticefficacy. Thermosensitive liposomes can further enhance tumor targetingand decrease toxicity by release of their water soluble drug contents inresponse to tumor hyperthermia. Various synthetic and natural lipidssuch as dipalmitoyl phosphatidyl choline and distearoyl phosphatidylcholine or egg phosphatidyl choline and cholesterol can be combined indifferent molar ratios with ethanol, or other agents that have abiphasic effect on gel-to-liquid phase transition of phosphatidylcholine bilayers, to produce liposomes that melt (undergo gel-to-liquidcrystalline phase transitions) at a predetermined hyperthermictemperature.

Thermosensitive liposomes were prepared form phosphatidyl choline (PC)and cholesterol (Ch) using the ethanol method of Tamura et al. Acombination of PC:Ch in a 8:1 molar ratio in the presence of 6% (v/v)ethanol resulted in formation of liposomes having a transitiontemperature between 40.2 and 40.8° C. The anticancer drug dacarbazine[5-(3,3′-dimethyl-1-triazino) imidazole-4-carboxamide] was encapsulatedin these heat-sensitive liposomes at a concentration of 3 mg/ml. The invivo efficacy of the thermosensitive, liposome encapsulated dacarbazinewas tested on Swiss albino mice transplanted with a dimethylbenzo-dithionaphtene derived ascites fibrosarcoma subjected to DNPinduced hyperthermia.

Male, 10-12-week-old, Swiss albino mice were injected with 3×10⁶ viablefibrosarcoma cells into the peritoneum. After 15 days the animals weredivided into various treatment and control groups receivingintraperitoneal injections of free dacarbazine, DNP alone, DNP+emptyliposomes and DNP+liposome encapsulated dacarbazine. DNP inducedhyperthermia was recorded with neonatal rectal and 22 ga. hypodermic YSIprobes. Temperatures were recorded 30 minutes after a 20 mg/kgintraperitoneal dose of DNP. DNP was administered every day for a totalof 5 doses. In all cases the hypodermic, intraperitoneal temperatureswere 1° C. higher than the rectal.

As shown in FIG. 20, survival curves of animals treated with DNP aloneand DNP+drug containing liposomes were significantly improved incomparison to controls. DNP-hyperthermia treated animals remained aliveat day 100 whereas sham treated animals all died by 60.

Example 6 Use of DNP to Induce Autologous Heat Shock Proteins as a Formof Thermal Preconditioning Prior to Arterial Balloon Catheterization orIschemic Surgical Injury

DNP would be given orally at doses to increase the V0₂ from 1.5 to 5times above normal per day for a period of 2-6 days or, as an infusionat doses that would increase V0₂ and core body temperatures no greaterthan 39° C. for periods of 5 to 6 hours or, intravenous doses of DNPalone, with vasopressors, or other short acting metabolic stimulators,that would increase V02 to equivalent core temperatures of 40-41° C. forperiods of 15-30 minutes. Within 8-48 hours after cessation of DNP, thepatient would have maximum heat shock protein production. Such DNPinduced stress would improve clinical outcome by induction of cellularheat shock protein synthesis with protection of the patient's, organs,tissues and cells from subsequent ischemic surgical or traumaticprocedures.

This method of DNP induced preconditioning could be used to decreaseintimal thickening and restenosis after angioplasty, improveischemia/reperfusion injury in organ and tissue transplantation, andimprove surgical outcome of procedures that require temporary orprolonged occlusion of arterial blood flow. Examples of such DNP inducedautologous thermotolerance used as a form of preconditioning aredepicted in FIG. 21, which shows limitation of proliferative arterialcatheter balloon injury in Sprague-Dawley rats pretreated with DNPinduced hyperthermia; FIG. 22 shows the protective effect of DNPpretreatment before hepatic ischemic injury cased by Pringle's maneuver;and, FIG. 23 depicts improved musculocutaneous flap skin survival afterinduction of heat shock proteins by DNP.

Example 7 Method of Using DNP to Enhance Proton Emission Tomography(PET) in the Diagnosis of Malignancy and/or Malignant Transformation(GLIOMA)

History: A 24 year old white male with neurofibromatosis presented witha six month history of left sided loss of body sensation, emotionalchanges, sensory seizures, inattention to conversations and sensationsof jamais vu.

Physical examination: weight=65 kg; height=175 cm; BP=135/80; HR=86 &reg; Resp=18; T=37.9° C. The patient was a well developed well nourishedwhite male with left upper and lower extremity sensory loss, posturalinstability and loss of tactile discrimination. There was a frank lefthanded astereognosis. Eye examination was normal, without papilledema.

Laboratory studies: Complete hemogram, blood chemistry and endocrineexamination were normal. EEG was within normal limits. MRI withgadolinium enhancement showed a decreased signal in the righttemporoparietal region with no evidence of contrast enhancement. PETexamination with [¹⁸F]fluoro-2-deoxy-D-glucose (FDG) revealed ahomogeneous hypometabolic area (metabolic Grade 1) consistent with a Lowgrade glioma in the right temporoparietal region. There were no zones ofhigh FDG uptake. Differentiation of displaced noninvaded gray matterfrom the tumor was not discernible on PET imaging.

Clinical assessment and diagnostic evaluation: although Low gradegliomas generally present histological features of benign tumor, it isknown that the presence of zones of high FDG uptake by PET scan in suchgliomas is associated with a higher percentage of malignanttransformation. PET-FDG with evidence of tumor hypermetabolism isbelieved to be an early biochemical marker of cellular malignanttransformation and is of prognostic value in High grade gliomas.Biochemically, high glucose (uptake of FDG) utilization in the presenceof oxygen, known as aerobic glycolysis, is believed to be the result ofa hyperactive hexokinase attached to tumor mitochondria. Increased FDGuptake therefore, represents increased hexokinase activity and isassociated with increased aggressiveness in gliomas, menigiomas andother neoplasms. Since DNP uncouples oxidative phosphorylation, anyshortfall in mitochondrial ATP production must come from increasedglycolysis. As a result, FDG uptake will be proportionately increased inDNP treated malignant cells over those that are normal in contralateralbrain white and gray matter. Since no abnormal FDG uptake was detectedin the tumor by standard PET methodology and the PET scan was unable toclearly delineate the borders of the tumor, it was elected to give thepatient a low dose of DNP to enhance FDG uptake and repeat the PET scan.Hypermetabolic components of the tumor would thus permit a more focusedPET-guided stereotactic biopsy.

Pretreatment protocol: three days prior to DNP dosing and repeat PET-FDGscan, the patient's dosage of phenyloin was increased from 100-mg threetimes daily to 200-mg three times a day. The same positron emissiontomogram, a CTI-Siemens 933/08-12 which provides a 6.75-mm adjacentslices and in-plane spatial resolution (full-width at half maximum) of˜5 mm, was to be used. The highest level of non to DNP stimulated FDGuptake in the tumor area was to be compared and qualitatively graded bytwo radiologists. Independently, each investigator was to visuallyevaluate the positron emission tomogram and use the following metabolicgrading scale: I, FDG uptake less than contralateral white matter; II,uptake between the levels in contralateral white and gray matter; III,FDG uptake equal to or greater than in contralateral gray matter.

Diagnostic—treatment protocol: the patient was given a 300 mg capsule ofDNP (approximately 4 mg/kg body weight) three hours prior to undergoinga PET-FDG scan. Forty minutes prior to the emission scan he wasintravenously injected with a bolus of FDG according to standardmethodology. Immediately prior to the 20-minute emission scan thepatients VO₂ uptake was 40% above that at baseline. The patients DNP/V0₂flow chart is FIG. 24.

Diagnostic outcome: DNP enhanced PET-FDG scan revealed two areas ofhypermetabolism. One of the areas surpassed the limits of the lesion onCT images and consequently only one of the targets (graded as a III onFDG uptake) was selected in the “abnormal PET-normal CT” area.

The plane that best displayed the abnormal FDG hypermetabolic uptakearea was selected and a pixel located in the center of the zone wasinteractively pointed at on visual inspection. The coordinates of thatDNP induced hypermetabolic pixel were then calculated and set as atarget for biopsy. A PET-guided stereotactic biopsy was performed underthe procedure described by Levivier et al., i.e., the target from thePET image was projected onto the corresponding stereotactic computedtomographic (CT) slice to control the reliability and precision oftarget selection and the trajectory. Serial stereotactic biopsies wereperformed along the trajectory by the method described by Kelly et al.

On pathologic examination, including analysis of nuclear polymorphismand cell density, 2 foci of anaplasia consistent with glioblastoma(Grade III astrocytoma) were noted.

Treatment outcome: based on the DNP enhanced PET-FDG scan diagnosticsoutline above, this patient was found to have a malignant transformationin his otherwise Low grade glioma. This diagnostic treatment protocolprocedure of detecting foci of hypermetabolism caused him to undergosystematic radiation therapy with chemotherapy(dibromodulcitol-procarbazine-carmustine) early in the course of hismalignant process. One year after diagnosis and therapy the patientagain underwent PET scanning DNP enhancement (repeated as outlined under“Diagnostic” above) revealed a single hypermetabolic component(metabolic Grade II) in the tumor area. Repeat PET-guided biopsyrevealed the area to be a zone of radionecrosis. The remaining viabletumor, even with DNP enhancement, continued to be a metabolic Grade I.The patient remains alive one and a half years after his diagnosis,albeit with left-sided hemiparesis.

Example 8 Method of Using DNP to Enhance Detection of Malignant Tumorsby High Resolution Digital Infrared Imaging (Breast Carclnoma)

History: a 34 year old white female with existing fibrocystic disease ofthe breast underwent yearly mammography and was found to have anequivocal opacity in the right breast, medial to the aereola. Two pastbreast biopsies were negative for malignancy and consistent withfibroadenomatous disease of the breast. The patient was opposed toanother breast biopsy (would be third), unless there was a definitiveindication of a lesion over that of her known fibrocystic disease of thebreasts.

Physical examination: WT=60 kg; HT=164 cm; BP=120/72; HR=88 & reg;R=18/min; T=37.7C. The patient was a normal appearing white female withscattered to diffuse nodularities in both breasts. A palpable 3×2 cm,non-tender, lump was located 3 cm medial to the right aereola. There wasabsence of nipple discharge, retraction, skin dimpling, rash ordiscoloration of either breast. There were no palpable axillarylymphadenopathy.

Laboratory studies: chest x-ray, EKG, blood chemistry, and hemogramexamination was normal. Mammography, Doppler ultrasound, MRI, andscintinammography failed to indicate or eliminate a possible occultcarcinoma in this young patient with dense, fibroadenomatous breastdisease. A diffuse, non-cystic, opacity on the right breast was the onlydefinitive finding from these breast studies.

Clinical assessment and diagnostic evaluation: this patient has had twoprevious open breast biopsies without evidence of malignancy. Earlydetection of breast carcinoma is of crucial importance to survival.False negative results of mammography (and other complimentary studies)range between 5-30%. The ability of infrared imaging technology todetect changes related to increased metabolism (tumor) and angiogenesishas greatly improved from that of 30 years ago. High resolution digitalcomputerized infrared equipment can now detect focal increases in tumortemperature from as little as 0.05° C., and increases in focal breasttemperatures may be as high as 1-2° C. in malignant tumors versusnormal, contralateral breast sites.

Since it is known that infrared imaging has at least a 19% rate of falsepositives and 17% of false negatives, and equivocal mammography andabnormal infrared imaging is not uncommon in young women with densebreast tissue and diffuse fibrocystic disease, the use of DNP to enhancetumor metabolism (infrared imaging) over that of normal tissue, could beof substantial diagnostic benefit. Specifically, DNP would greatlyenhance tumor metabolism (infrared imaging), in comparison to non-DNPenhanced infrared imaging and would greatly increase tumor detectionwhen there is either insufficient production or detection of metabolicheat or vascular changes. Further, the heat differential between DNPenhanced and non-DNP infrared tumor imaging may also decrease the falsepositive rate seen with this procedure, especially in benign conditionssuch as fibrocystic disease of the breast. Since non-DNP infraredimaging is capable of detecting as great as 1-3° C. changes in focaltemperature between normal and malignant tissue, DNP enhancement wouldincrease the temperature difference several fold and enhance both thesensitivity and precision of currently available infrared imagingtechnology. The patient agreed to have both of her breasts examinednon-invasively with infrared imaging, before and after intravenous DNPadministration to ascertain if there was increased infrared signalingfrom the worrisome, palpable lump in her right breast.

Prediagnostic protocol: the patient was disrobed to the waist and satwith her hands interlocked over her head for a five minute equilibrationperiod in a draft free, thermally controlled room—kept between 18° C.and 20° C. She did not take any oral medication, alcohol, coffee, anddid not smoke, exercise or use deodorant three hours prior to testing. Abaseline of 4 images consisting of an anterior, undersurface and 2lateral views of each breast were generated by an integrated infraredimaging station consisting of a scanning mirror optical systemcontaining a mercury-cadmium-telleride detector (Bales Scientific, CA).The infrared system had a spatial resolution of 600 optical lines, acentral computerized software processor providing multi-taskingcapabilities and a high-resolution color monitor capable of displaying1024×768 resolution points with 110 colors or shades of gray per image.Images were stored on retrievable laser discs.

Diagnostic treatment protocol: after the above baseline studies wereperformed, the patient was given an initial intravenous dose of 1 mg/kgDNP and observed for a period of 20 minutes. An additional 2 mg/kg ofDNP was then administered and 30 minutes thereafter, she was taken tothe thermally controlled room for repeat DNP-enhanced infrared imaging.Immediately prior to transferring the patient to the thermallycontrolled room, the patients VO₂ was incrementally increased to 50%above her VO₂ baseline, see FIG. 25. Repeat infrared images were thenobtained under the exact protocol used for obtaining baseline studies.

Diagnostic—treatment outcome: baseline (non-DNP enhanced) infraredimaging revealed insignificant vascular asymmetry and no significanttemperature changes when the results were reviewed and compared to therest of the ipsilateral or contralateral breast sites.

DNP enhanced infrared imaging resulted in a bilateral global breasttemperature increase of approximately 0.5° C. An abnormal, 2.5° C.increase in temperature was noted in the palpable, right breast lesiondiscovered by clinical exam. Since no non-cancer causes for such adramatic temperature increase could be identified, i.e. abcess, trauma,or recent surgery, this 5 fold increase in heat production (above theDNP baseline increase of 0.5° C.) was highly suspect to be caused by anearly malignancy.

The patient was admitted to the hospital and under general anesthesiaunderwent an open breast biopsy. Frozen section (and later permanenttissue mounts) revealed a well-differentiated intraductal carcinoma.Progesterone and estrogen receptors, as determined by immunocytochemicalmethods, were negative. A simple, right mastectomy with axillary lymphnode dissection was performed. A total of twelve lymph nodes wereidentified: there was no evidence of tumor. The patient refusedchemotherapy and radiotherapy. She was placed on long-term oraltamoxifen (10 mg twice a day).

Example 9 The Use of Dinitrophenol with Artificial Electron Receptors(or Other Free Radical Forming Agents) in the Treatment of Hormone andChemotherapy Resistant Malignancy (Prostate Cancer)

History: a 68 year old Mexican male, developed a gradual increase in lowback pain, right hip pain and several episodes of hematuria over a 10month period. He was referred to a urologist and diagnostic work-uprevealed a carcinoma of the prostate with the extension of the tumorinto the bladder. Bony metastasis were present to the right pelvis,fourth and fifth lumbar vertebra, right femur, left humerus, right sixthand seventh ribs and right scapula. He refused any form of surgery butunderwent radiation therapy to the pelvis and symptomatic bony lesions.Treatment was initiated with megestrol acetate (640 mg/day), prednisone(20 mg/day) and leuprolide (7.5 mg/month). After three months of therapythe patient continued to have progression of his disease manifested byincreasing bone pain, rising prostatic specific antigen levels (PSA) andincreasing serum acid phosphatase.

Physical examination: WT=72 kg; HT=175 cm; BP=140/86; R=22; T=37.6 C;HR=88 & reg; Exam revealed mild emaciation with some scrotal and +1pitting bilateral lower extremity edema. There were scattered bilateral,basilar rales on examination of the chest.

Laboratory studies: EKG demonstrated a right partial bundle branchblock. Chest x-ray showed mild chronic obstructive pulmonary diseasewith minimal fibrosis. There was some patchy, interstitial edema in bothlower lung fields. There were no pulmonary metastasis. Complete bloodcount showed a mild anemia with a hemoglobin of 10.5 and a hematocrit of34%. Liver function tests were normal. White blood cell count,differential and platelet count, was within normal limits. PSA level was58 ng/ml. Serum acid phosphatase was 2× above normal. Blood electrolytesincluding calcium were within normal limits. The acid phosphatase, AST,ALT and bilirubin levels were normal. Radionucleotide bone scan revealedmultiple metastasis in the axial skeleton and ribs. Review of pastprostatic biopsy slides showed a high grade adenocarcinoma of theprostate with a Gleason Grade of 8. Pulmonary function studies showedmoderate airflow obstruction with mild hypoxemia and hypercarbia. StressEKG was not performed because of his severe exercise intolerance.

Clinical assessment and treatment evaluation: the patient has ametastatic, hormone-refractory prostate carcinoma with clinicalprogression documented by increasing bone pain and rising serial PSAvalues. Under the TNM classification of the American Joint CancerCommittee for prostate cancer (T=degree of primary tumor extension;N=regional lymph node involvement; and, M=presence of distantmetastasis), he has the highest stage (T4 N3 M1). Histologically, thetumor is aggressive by the Gleason Grading System. Since death due toprostatic carcinoma is almost invariably a result of failure to controlmetastatic disease, and since prostatic cancers are well-known to besensitive to heat stress, the present DNP therapy was undertaken as alast resort effort to stop tumor progression and/or improve the patientsquality of life.

In view of the patients age, pulmonary problems and poor performancestatus (Karnofsky Score of 6) it was decided to treat the patient withmoderate doses of DNP and a free radical cycling agent, methylene blue(MB), to induce synergistic tumor killing. The effect of methylene blueon cellular reduction-oxidation status (redox) is well known. Methyleneblue readily traverses cell membranes and acts as an electron acceptorfrom the major coenzymes. Unlike other oxidizing drugs, it cyclesfutilely, transferring electrons from endogenous substrates to oxygen.Depending on the redox status of a cell, MB can act as either anintracellular electron acceptor or donor. MB directly catalyzes thereaction of intracellular reductants, NADPH, NADH and GSH (reducedglutathione) with oxygen causing the production of hydrogen peroxide,superoxide anions, and the formation of the potent cytotoxic oxidantspecies, peroxynitrite. In DNP partially uncoupled mitochondria, MBfurther stimulates respiration due to its dual action of providingreducing equivalents necessary for beta-oxidation of fats and electrondonating/shuttling capacity, with respect to the mitochondrialrespiratory chain. It is an effective drug, at doses of 1-3 mg/kg, intreating nitrate-induced methemoglobinemia. MB is also used as anantidote given as a 100 mg IV bolus for encephalopathy associated withalkylating chemotherapy.

Since uncoupling, heat and MB increase the flux of cellular freeradicals and malignant cells possess a high bioreductive capacity, thesynergistic effects of DNP with MB would allow for maximum tumor killingwith minimum to moderate levels of induced total body hyperthermia.Additional free radical cycling agents that can be used in lieu of MBinclude, but are not limited to: phenazine methosulfate, xenobioticssuch as quinones (e.g., menadione, semiquinone, naphthoquinone,duroquinone, indigo carmine), nitrocompounds (e.g., metronidazole,niridazole, nitrofurazone, flunitrazepam), eminium ions (e.g., methylviologen, benzyl viologen, etc.), and others. In this patient, DNP-MBtherapy was to be administered so as not to exceed the baseline VO₂level by 50-75%.

Pretreatment protocol: the patient was transfused with 2 units of packedred blood cells 48 hours prior to undergoing treatment. Intravenousfluids (Lactated Ringer's solution) were administered at a rate of 100cc/hour. The patient was dressed in comfortable cotton clothing andplaced in an air-conditioned room. Equipment for monitoring heart rateand rhythm, temperature and oxygen consumption was utilized as outlinedin Example 1. An oral breathing tube was used to conduct TEEM VO₂measurements. Oxygen supplementation and “crash cart” was available atbedside.

Treatment protocol: baseline VO₂ measurements for 8 minutes establishedan average VO₂ of 250 cc/minute. DNP, at a dose of 2 mg/kg, was infusedintravenously over a 2 minute period. Repeat VO₂ at 20 minutes wasstabilized at 340-360 cc/minute. An additional 1 mg/kg DNP infusion wasadministered, and 15 minutes thereafter the VO₂ increased and stabilizedat 420 cc/minute. Ten minutes thereafter, an infusion of methylene blue,2 mg/kg (dissolved in a 0.4% pyrogen-free isotonic saline solution-35ml) was administered over 20 minutes. Repeat VO₂ measurement at 20minute intervals showed it to rise to and stabilize at 450-500cc/minute.

By hour 3, VO₂ declined to the 360-380 cc/minute range. An additional 1mg/kg dose of DNP was infused over a 2 minute period. Repeat VO₂measurements 20 minutes after this infusion showed an increase in VO₂back to the 450-500 cc/minute. Rectal probe temperature increased to amaximum of 1.3° C. over baseline. Blood pressure and cardiac ratesremained within normal limits. The patient withstood the procedurewithout any adverse effects and therapy was terminated 6 hours after theinitial DNP dose. The protocol was repeated every other day for a totalof 15 treatments (30 days). Therapy was discontinued for 2 weeks and thecycle was again repeated for an additional 30 days, treatment beingadministered every other day.

Treatment outcome: there was no evidence of general toxicity at any timeduring treatment. The patient noted a decrease in his low back, hip andother areas of bone pain on the 6^(th) day following therapy. By 2weeks, the patient was off all narcotic (morphine) analgesics and had amarkedly increased appetite. On day 8, repeat PSA levels were increasedby approximately 120% to 125 ng/ml. Acid phosphatase remained unchanged.All other blood chemistries, including CBC, showed no significantalterations.

At 6 weeks after treatment, repeat PSA values showed a significantdecline to 30 ng/ml with a concomitant fall in serum acid phosphataselevels. At the final stage, 10 weeks after initiation of treatment, aprostatic biopsy was performed. Histologic examination revealed 95% ofthe tumor to be necrotic with only scattered or scarred acini containingan occasional malignant cell. There was a significant increase instromal cells above that seen in his initial biopsy. One of the moststriking changes noted by the pathologist was the formation of cyst-likestructures within the epithelial cells. The patient was seen threemonths after initiation of therapy, at which time he had gained 8.2 kgof weight, remained pain free and stated that he felt “normal”. FIG. 26shows monitored treatment parameters. FIG. 27 shows biochemical, biopsyand clinical responses.

Oral DNP therapy (250 mg twice a day, daily for 5 days and recycledafter no medication for 2 days) was initiated after his IV therapy andcontinued up to 4 months. A repeat prostate biopsy at the end of month 4was obtained. Pathologic examination revealed disintegration ofremaining tumor acini along with the formation of with many epithelialcysts. Occasional residual tumor cells were fractured and disrupted withmarkedly reduced cytoplasm. There was extensive fibrosis with anapparent increase in the number of stromal cells. Cytoplasm volume wassignificantly diminished in both the residual tumor and normal cells.Overall, there were very few intact acini or viable acinar cells.

Example 10 Method of Using Dinitrophenol with Biologic ResponseModifiers (in the Treatment of Hepatitis C Infection)

History: a 32 year old Investment Banker was evaluated for chronicHepatitis C infection. She gave a past history of intermittent jaundice,dark urine, mild anorexia, nausea and vomiting. This episode occurred 10years ago, approximately 3 months after a transfusion (3 units of packedred blood cells) for a cesarean section. She was currently asymptomatic,but on routine health insurance exam she was found to have elevations inher ALT and AST (alanine and aspartate aminotransferase) levels: 140IU/L and 90 IU/L, respectively. She drank 5-8 glasses of wine per week.Additional laboratory tests identified anti-HCV antibodies with anHCV-RNA level of 5×10⁶/ml. The patient refused to undergo liver biopsybut agreed to treatment with interferon alpha-2b (3 million unitsinjected subcutaneously 3 times per week) and ribavirin (500 mgorally-twice a day). After 12 weeks of treatment she developed lethargy,severe headaches, fever, nausea and depression. Anemia was detected witha hemoglobin concentration of 9.2 g/deciliter. As a result, her dosageof interferon was reduced to 1.5 million units 3 times a week and thedose of ribavirin was reduced to a total of 600 mg/daily. After 6 monthsof treatment her ALT and AST levels became normal and HCV-RNA becameundetectable.

An additional six months of therapy however, failed to sustain herclinical improvement and she was found to have a relapse. Serum HCV-RNAlevels rose to 5.2 million copies/ml and liver enzymes increased to2.5-3 times that of the normal range. She was unable to tolerate anyadditional ribavirin because of severe anemia. She persistently refusedto undergo a percutaneous liver biopsy.

Physical examination: WT=48 kg; HT=150 cm; BP=128/82; HR=76 & reg; R=18;T=37.5° C. Physical examination failed to reveal any signs of chronicliver disease. She was noted to have several scattered areas of scalpalopecia which she attributed to her anti-hepatitis C therapy.

Laboratory studies: EKG and chest x-ray were normal. CBC revealed a mildanemia with a hemoglobin of 10.2 and a hematocrit of 34%. WBC,differential and platelet count were within normal limits. Alkalinephosphatase was within normal limits. Serum AST and ALT were elevated to2.5-3 times that of the upper normal limit. Serum HCV-RNA levels werefound to be at 5.8 million copies/ml. The infecting hepatitis C strainwas of genotype 1b. Antimitochondrial antibody serology was negative(titer less than 1:20). There were no other blood chemistry, hormone, orurine laboratory abnormalities.

Clinical assessment and treatment evaluation: the patient has a chronicHepatitis C infection with relapse after combination ribavirin andinterferon alpha-2b treatment. This is not uncommon in that the rate ofrelapse after an end-of-treatment response to interferon-ribavirintherapy may exceed 50%. She was unable to tolerate additional ribavirintherapy because of a related anemia. Further, interferon dose escalationin non-responders to initial interferon therapy has only provedsuccessful in a small number of cases. Despite her refusal to undergoany form of liver biopsy she agreed to undergo a combination of DNP andinterferon therapy for a period of 12 weeks.

The liver is known to be one of the “hottest” organs in the human body.Liver temperatures exceeding 44° C. have been documented in humansundergoing strenuous exercise. The hepatitis C virus is an RNA encodedsphere containing several polyproteins comprising a capsid, 2 envelopeproteins, and at least 6 enzymatic proteins with varied functions.Hepatitis C virus is known to be heat sensitive and is inactivated bystandard blood banking heating techniques. Case reports of hepatitis Cinactivation with the use of extracorporeal hyperthermia are known. Ithas been reported that HIV positive patients treated with extracorporealhyperthermia, many of which were also positive for hepatitis C, thehepatitis C virus was cleared (as determined by serum viral PCR-RNAanalysis).

Based on the this patients failure to respond to conventional treatment,anecdotal and case report studies showing beneficial results with wholebody hyperthermia, the patient underwent a combination of DNP andinterferon therapy. She was informed that she would undergo dailytreatments with intravenous DNP for five days per week and receiveinterferon alpha at a dose of 1.5 million units subcutaneously every twodays. This treatment protocol would continue until her hepatitis C-RNAblood viremia was no longer detectable.

Pretreatment protocol: each evening prior to treatment the patient wasinstructed not to eat after 7 pm and dress in cotton clothes.Approximately 6 hours prior to intravenous DNP administration she was tobe given 1.5 million units of subcutaneous interferon-alpha every 3rdday. Repeat blood work, including CBC and platelet count, AST, ALT, andhepatitis C-RNA levels would be initially obtained at 48 hours andweekly thereafter. No efforts were to be made to prevent body heat loss.A single intravenous line was placed with a 21-gauge interacath. Heartrate, rectal thermistor, and VO₂ monitoring was conducted during therapyas outlined.

Treatment procedure: the patient presented herself for outpatienttreatment and was given a subcutaneous dose of 1.5 million units ofinterferon-alpha. Approximately 6 hours thereafter, at 1 pm, a baselineVO₂ recording of 5 minutes was 160 cc/min. She was infused with 1 mg/kgDNP over a 2 minute period. At 20 minutes, her VO₂ increased andstabilized at approximately 210 cc/min. A second dose of 1 mg/kg DNP wasinfused and the VO₂ peaked 20 minutes later at 250 cc/min. An additionaldose of 2.0 mg/kg DNP was given 30 minutes following the second dose.Repeat VO₂ showed a rise and stabilization 20 minutes thereafter at 360cc/min. The patient's rectal temperature increased and never exceeded1.3° C. above her normal baseline. Two hours after her last dose of DNP,her VO₂ declined to 280 cc/min. An additional 2 mg/kg dose of DNP wasadministered. The patients VO₂ increased and stabilized 20 minutesthereafter to a level of 420 cc/min. She was noted to sweat profusely.Throughout treatment the patient was permitted to drink fluids adlibitum. She was notably fatigued at hour 5 of therapy. Monitoredparameters and flow chart are shown in FIG. 23. The 5 day treatmentprotocol was repeated after a 2 day “DNP rest period”. This regimen wasrepeated times 3. Subcutaneous interferon-alpha was administered for atotal of 10 weeks. FIG. 28 shows the patients DNP/interferon treatmentflow chart.

Treatment outcome: by the treatment regimen outlined above, hepatitisC-RNA viral load decreased by approximately 2 logs after 48 hours. Overthe next 5 days the viral load further decreased by an additional log.HCV-RNA became undetectable and the HCV viral genome remained clearedfrom the bloodstream at week 2 and thereafter. Alanine transaminase(ALT) levels increased 7 fold at 48 hours and remained elevated untilweek 3, at which time they returned to levels slightly below that whichexisted prior to therapy. CBC, bilirubin, and blood urea nitrogen (BUN)remained within normal limits. Alkaline phosphatase levels increased 2fold at 48 hours but returned to pretreatment levels at day 7.

The patients HCV viral genome remained cleared from her bloodstream 18months after therapy and there was normalization of her ALT.

Example 11 Method of Using Dinitrophenol Induced IntracellularHyperthermia to Increase Immunogenicity of Human Tumors

DNP would be given as an intravenous solution, or as an oralpreparation, so as to increase oxygen consumption 2.5-5 times abovenormal for a period of 2-3 hours. Such treatment would be administeredevery other day for a period of 5-10 days. At 8-24 hours after the lastday of treatment, the patient would be administered standardchemotherapy or specific monoclonal antibody immunotherapy directedagainst known mutated or inappropriately expressed oncogenic proteins(e.g., ras, p53, HER/neu, etc.), or combination anti-oncogenicimmunotherapy with chemotherapy or radiation.

Heat shock proteins (HSPs) or stress-induced proteins are constitutivelyexpressed in all living cells and are among the most abundant proteinsfound. However, many members of the HSP family can further be expressedby cellular stress-causing conditions such as heat, drugs, glucosedeprivation, etc. Of importance to the present method is that theexpression of HSPs in tumors is associated with a heightened immuneand/or cytotoxic T-lymphocyte response. In particular, it is known thatmembers of the HSP70 family (HSPs are generally classified by theirmolecular weights e.g., HSP90 kdaltons, HSP27 kdaltons, HSP70 kdaltons,etc.) are expressed on cell surfaces. Due to the ability of DNP toinduce intracellular hyperthermia, the enhanced expression of human HSPsin DNP treated tumors could greatly increase their immunogenicity.

This method could be used to broaden the antigen-specific repertoire ofmany poorly immunogenic tumors by increasing the expression ofHSP-peptide immunogenic determinants on their cell surfaces. Suchconsequences would heighten any endogenous specific anti-tumor immuneresponse. Moreover, DNP-intracellular heat-inducible immunogenic targetscould further increase the efficacy of exogenously synthesized andadministered monoclonal antibodies. By example, patients with HER-2/neuoverexpressing metastatic breast cancer (25% of breast cancer patients)would be treated by the DNP method outlined above. This treatment wouldthen be followed by a standard loading dose and weekly infusions ofanti-HER-2/neu monoclonal antibodies. Clinical benefits would beevaluated by overall response rates and duration of response.

Example 12 Synthesis and Use of Novel Conjugates and Derivatives of2,4-Dinitrophenol

Formation of novel nitrophenol compounds is of importance in that theiralkyl, alkene, fatty acid, aromatic and other derivatives maysignificantly enhance their biologic activity and/or improve thetherapeutic index. Many reactions of the benzene ring of phenols throughhalogenation, sulfonation, and nitration are known. Numerous proceduresfor C-alkylation of phenols through reduction of benzylic alcohol,aldehydes, benzonitriles and Mannich bases are published.

Alkylations or other “R” group additions have also been performed onvarious phenolic substrates using Stille or Negishi coupling reactions.An example of converting a nitrophenol compound to the desired alkylated(or other “R” group analog) by a 2 step procedure utilizing the Stillecoupling reaction is illustrated in FIG. 30. As shown in step 1, DNP isfirst iodinated with Barluenga's reagent (IPy₂ BF₄) to yield2,4-dinitro-3,5-diiodophenol. In step 2, the nitroiodophenol is thenconverted to the alkylated derivative (in the instant example anethylated derivative) via a co-catalytic, palladium-copper Stillereaction.

Compound 3 shown in FIG. 30 is an ethylated derivative of DNP and isdesigned to increase uncoupling activity by adding lipophylic alkylsubstituents to the benzene ring. Such analogs with augmented activitymay be particularly useful in the treatment of bulky tumors andmalignancies which possess a high fat content, e.g. liposarcoma,glioblastoma, etc.

A representative approach (Step 2) to the palladium-copper, co-catalyticethylation of a nitroiodophenol is illustrated by the conversion of2,4-dinitro-3,5-diiodophenol to 2,4-dinitro-3,5-diethylophenol.Nitroiodophenol (500 mg, 934 μmol) is added to a pressurized reaction tocontaining N-ethylpyrrolidinone (1.5 ml). Pd₂ dba₃CHCL₃ (27 mg, 26 μmol)and triphenylphosphine (50 mg, 191 μmol) is added to the stirringsolution and slowly heated to approximately 50° C. for 10 minutes.Copper iodide (17 mg, 91 μmol) is added to the stirring solution. Themixture is again heated to 50° C. for 10 minutes. The solution is cooledto 32° C. and tetraethyl tin (285 μL, 2.05 mmol) is added to thestirring solution. The reaction tube is sealed and heated withcontinuous stirring at 65° C. for 12-16 hours. Aqueous workup and ethylacetate extraction with drying by magnesium sulfate (MgSO₄) andconcentration yields the end product.

Example 13 Synthesis of an Expanded Combinatorial Library of PutativeUncoupling Agents Capable of Inducing Intracellular Hyperthermia

The spectrum of potential classic uncoupling agents that can induceintracellular hyperthermia can be greatly expanded through a designedconvergent synthetic approach. An almost limitless variety of uncouplerscan be synthesized through a “combinatorialized” scheme to produce anexpanded “library” of uncoupling agents with related structures. Thescheme specifically presented herein exemplifies the synthesis of 21potential uncoupling agents, but can be expanded to 1,000 to 100,000putative uncoupling agents.

Five classes of uncouplers are prepared via the convergent route shownin FIG. 31. The synthetic scheme depicted in FIG. 31 is designed as acombinatorial approach to allow access to a library of structurallyrelated putative uncouplers for biological evaluation. While the givenexamples noted in FIG. 31 will allow formation of at least 21 noveluncouplers, a simple variation in this synthetic scheme will allow thelibrary of uncouplers to be expanded to include from 1,000 to 100,000novel uncoupling agents. After discussing the general synthetic approachin FIG. 31, the simple synthetic variations designed to expand thelibrary of uncouplers will be described. Such variations will beapparent to those skilled in the art of synthetic organic chemistry andpharmaceutical development.

Starting from benzaldehyde (FIG. 31, Compound 1), diiodination at the 3-and 5-positions using Barluenga's reagent (IPy₂BF₄) affords Compound 2which is alkylated using a co-catalytic, palladium-copper Stillereaction to produce a 3,5-disubstituted Compound 3. This 2 step approachis known for producing a variety of methylated phenols. Use oftetramethyltin then produces the dimethyl derivatives [Compound 3, whereR=Me(methyl)]; tetrabutyltin produces the dibutyl derivatives [Compound3, where R=Bu(butyl)]; and, tetraphenyltin produces the diphenylderivatives [Compound 3, where R=Ph(phenyl)]. A Baeyer-Villigeroxidation of Compound 3, with meta-chlorobenzoic peracid (mCPBA)followed by alkaline hydrolysis [KOH (potassium hydroxide)] of theresulting formate affords phenols, Compound 4. The homogeneous2,4-dinitro-or 2,4-dicyano-derivatives are initially accessed from anintermediate Compound 4. Nitrosation of Compound 4 withnitrofluoromethylsulfonate salt (NO₂CF₃SO₃) provides the3,5-disubstituted-2,4-dinitrophenols shown in Compound 5. Threedifferent uncoupling agents are produced via this synthetic route.Diiodination of Compound 4 at the 1- and 4-positions produces Compound 6which is treated with copper(I)cyanide (CuCN) to give the 2,6-dicyanatederivative, Compound 7. Three additional uncouplers are synthesized bythis route. The heterogeneous nitro-, cyano-uncouplers are also accessedfrom intermediate Compound 3. The 2-cyano-, 4-nitro-uncouplers aretargeted as the steric effects of the cyano group at the 2-position isless than the corresponding 2-nitro-derivatives. Mono-iodination ofCompound 3 through the thallium intermediate affords the selective2-iodo-derivative, Compound 8. Conversion of Compound 8 to phenol,Compound 9, is accomplished as before through the Baeyer-Villigeroxidation and hydrolysis of the resulting formate. Selective 4-nitrationto produce Compound 10 is readily accomplished withnitrotrifluoromethylsulfonate salt followed by cyanation to afford2-cyano-,4-nitro-uncouplers, Compound 11. Three additional uncouplersare produced by this route.

Additional uncouplers, such as the 2,4,6-tricyano compounds, can also beproduced through the same convergent synthesis. Exhaustive iodination ofCompound 4 affords 2,4,6-triiodinated Compound 12 which is then directlyconverted to tricyano-uncouplers, Compound 13, through copper catalyzedexchange. Three more uncouplers are produced by this modification. A2,4-dicyano-uncoupler carrying three variable substituents at the 3-, 5-and 6-positions is also readily produced through this convergentapproach. Initial selective monobromination of the phenol Compound 4 atthe ortho-position affords Compound 14 which is diiodinated at the2,4-positions to produce the 2,4-diiodo-, 6-bromo-Compound 15derivatives. Selective cyano exchange at the more reactive aryliodidepositions affords the dicyano Compound 16 derivatives. A finalco-catalytic, palladium-copper Stille reaction results in the formationof the 3,5,6-trisubstituted, 2,4,-dicyano-uncouplers. Use of the sametin reagents previously described allows the introduction of eithermethyl, ethyl, propyl, butyl, etc., or phenyl at the 6-position. Inconjunction with the 3 different substituents at the 3- and 5-positions,9 additional uncouplers are afforded by this additional expansive route.

The synthesis of 21 novel uncouplers depicted by the convergent approachin FIG. 31 can be further modified. To those skilled in the art, asimple variation in this exemplary synthetic approach will allow agreatly expanded library of potential uncouplers to be synthesized. Theexpanded library can be produced by introduction of an array of alkyland aryl substituents at the 3-, 5-, and/or 6-positions whilemaintaining the 2,4-dinitro-, 2,4-dicyano, 2-cyano-4-nitro-, and/or the2,4,6-tricyano-phenol substrate. This simple synthetic variation isaccomplished by using a variety of well known palladium, zinc, orcopper-mediated reactions at the stage of akly or aryl groupincorporation, i.e., FIG. 31, Compound 2 to 3 and Compound 16 to 17conversions. This synthesis is a variation on the Stille reaction, theHeck reaction, the Negishi coupling, Suzuki couplings, Semmelhackreactions and cuprate reactions. Such a variation can introduce a nearlyof unlimited array of potential substituents onto the phenol core of theuncoupler. This combinatorial approach can even be further expanded byvariable halogenation (either bromination or iodination) at the 3- and5-positions to allow 2 different substituents to be introduced at these2 positions in the ensuing metal-mediated halogen exchange reactions.This “combinatorial library” approach will allow a broad range ofpotential uncouplers to be synthesized and evaluated for potentialbioligical activity, including safety and effectiveness. Activity of themany diverse conjugates and derivatives of 2,4-dinitrophenol (and otheruncoupling agents) may be tested by known in vitro methods for oxygenconsumption, e.g., tissue or cellular suspensions with Clark type oxygensensors. Toxicity, mutagenicity and LD50 studies in animals would beperformed under recognized protocols prior to use of any such novelcompounds in human subjects. Upon establishing toxicity and safetycriteria, the various novel conjugates and derivatives can beadministered under dose escalation trials as outlined previously for theclinical use of dinitrophenol.

It will be apparent to those skilled in the art that numerousmodifications and variations can be made to the processes andcompositions of this invention. Thus, it is intended that the presentinvention cover the modifications and variations of this inventionprovided they come within the scope of the appended claims and theirequivalents.

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What is claimed is:
 1. A method for inducing intracellular hyperthermiacomprising the step of administering an amount of a mitochondrialuncoupling agent sufficient to induce intracellular hyperthermia.
 2. Themethod of claim 1, wherein the mitochondrial uncoupling agent is 2,4dinitrophenol.
 3. The method of claim 1, wherein the mitochondrialuncoupling agent is selected from the group consisting of: classicuncouplers, including 2,4 dinitrophenol, clofazimine, albendazole,cambendazole, oxibendazole, triclabendazole (TCZ),6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole and theirsulfoxide and sulfone metabolites, thiobendazole, rafoxanide, bithionol,niclosamide, eutypine, various lichen acids (hydroxybenzoic acids) suchas (+)usnic acid, vulpinic acid and atranorin,2′,5-dichloro-3-t-butyl-4′-nitrosalicylanilide (S-13),3,4′,5-trichlorosalicylanilide (DCC), platanetin,2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799,AU-1421,3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradecin-1,7(8H)-dione(zearalenone), N,N′-bis-(4-trifluoromethylphenyl)-urea, resorcylic acidlactones and their derivatives,3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847),2,2,-bis(hexafluoroacetonyl)acetone, triphenyl boron, carbonylcyanide4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA), carbonylcyanide 3-chlorophenylhydrazone (CICCP), 1,3,6,8-tetranitrocarbazole,tetrachlorobenzotriazole, 4-iso-octyl-2,6-dinitrophenol(Octyl-DNP),4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxalbisguanylhydrazone), pentachlorophenol (PCP),5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI),N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid),4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol,2-azido-4-nitrophenol, 5-nitrobenzotriazole,5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole,methyl-o-phenylhydrazone, N-phenylanthranilic acid,N-(3-nitrophenyl)anthranilic acid, N-(2,3-dimethylphenyl)anthranilicacid, mefenamic acid, diflunisal, flufenamix acid, N-(3-chlorophenyl)anthranilic acid, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone(FCCP), SR-4233 (Tirapazamine), atovaquone, carbonyl cyanide4-(6′-methyl-2′-benzothiazyl)-phenylhydrazone (BT-CCP), ellipticine,olivacine, ellipticinium, isoellipticine and related isomers,methyl-O-phenylhydrazonocyanoaceticacid,methyl-O-(3-chlorophenylhydrazono)cyanoacetic acid,2-(3′-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic acid,2-(2′,4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile,relanium, melipramine, and other diverse chemical entities includingunsaturated fatty acids (up to C₁₄ optimum), sulflaramid and itsmetabolite perfluorooctane sulfonamide (DESFA), perfluorooctanoate,clofibrate, Wy-14, 643, ciprofibrate, and fluoroalcohols; ionophorousantibiotic uncouplers, including gramicidin, nigericin, tyrothricin,tyrocidin, valinomycin, alamethicins, harzianin HA V, satumisporin SAIV, zervamicins, magainin, cecropins, melittin, hypelcins,suzukacillins, monensins, trichotoxins, antiamoebins, crystal violet,cyanine dyes, cadmium ion, trichosporin-B and their derivatives; andother heterogeneous coupling compounds, including desaspidin, ionizedcalcium (Ca⁺⁺), uncoupling proteins such as UCPI-1, UCP-2, UCP-3, PUMP(Plant Uncoupling Mitochondrial Protein), histones, polylysines,A206668-a protein, and compound K23187.
 4. The method of claim 1,wherein the mitochondrial uncoupling agent is a conjugate comprising 2,4dinitrophenol.
 5. The method of claim 1, further wherein the inducedintracellular hyperthermia is used in the diagnosis or treatment ofinfections, malignancies or other medical conditions.
 6. The method ofclaim 5, wherein the induced intracellular hyperthermia is used in thediagnosis or treatment of infections, malignancies or other medicalconditions selected from the group consisting of cancer, and infectionsor infestations.
 7. The method of claim 5, wherein the inducedintracellular hyperthermia is used in the diagnosis or treatment ofcancer.
 8. The method of claim 5, wherein an animal is administered themitochondrial uncoupling agent and a separate medication isadministered, wherein the second medication increases the overallmetabolic rate of the animal, the metabolic rate of a specific targettissue in the animal, or an increase in free radical flux.
 9. The methodof claim 8, wherein the second medication is selected from the groupconsisting of glucagon, arbutamine, dobutamine, vasopressin, glutamine,proline, octanoate, methylene blue (tetramethylthionine), ubiquinone,menadione, hematoprophyrin, polyunsaturated fatty acids includinglinoleic (double bonds at carbons 9 and 12), alpha-linolenic (doublebonds at carbons 9, 12, and 15), gamma-linolenic (double bonds atcarbons 6, 9, and 12), arachidonic (double bonds at carbons 5, 8, 11,and 14), eicosapentaenoic (double bonds at 5, 8, 11, 14, and 17),docosahexenoic (double bonds at carbons 4, 7, 10, 13, 16, and 19),cis-parinaric (double bonds at 9, 11, 13, and 15) and, monounsaturatedfatty acids including oleic (double bond at carbon 9), erucic (doublebond at carbon 13), phenazine methosulfate,2,6-dichlorophenolindophenol, coenzyme Q1, CoQ2 and their analogsduroquinone and decylubiquinone.
 10. The method of claim 5, wherein theinduced intracellular hyperthermia involve the induction of heat shockproteins.
 11. The method of claim 5, a second therapeutic agent, ortherapy, is administered.
 12. The method of claim 11, wherein thesecond, therapeutic agent or therapy, is selected from the groupconsisting of: anti-fungal agents, including Amphotericin B,Griseofulvin, Fluconazole (Diflucan), Intraconazole, 5 fluoro-cytosine(Flutocytosine, 5-FC), Ketatoconazole and Miconazole; anti-bacterialagents, including beta lactam rings (penicillins), macrocyclic lactonerings (macrolides), polycyclic derivatives of napthacenecarboxamide(tetracyclines), amino sugars in glycosidic linkages (aminoglycosides),peptides (bacitracin, gramicedin, polymixins, etc.), nitrobenzenederivatives of dichloroacedic acid, large ring compounds with conjugateddouble bond systems (polyenes), various sulfa drugs including thosederived from sulfanilamide (sulfonamides, 5-nitro-2-furianyl compounds(nitrofurans), quinolone carboxylic acids (nalidixic acid), fluorinatedquinilones (ciprofloxan, enoxacin, ofloxacin, etc.), nitroimidazoles(metroindazole), peptide antibiotics (such as bacitracin, bleomycin,cactinomycin, capreomycin, colistin, dactinomycin, gramacidin A,enduracitin, amphomycin, gramicidin J, mikamycins, polymyxins,stendomycin, actinomycin; aminoglycosides represented by streptomycin,neomycin, paromycin, gentamycin ribostamycin, tobramycin, amikacin;lividomycin beta lactams represented by benzylpenicillin, methicillin,oxacillin, hetacillin, piperacillin, amoxicillin and carbenacillin;lincosaminides represented by clindamycin, lincomycin, celesticetin,desalicetin; chloramphenicol; macrolides represented by erythromycins,lankamycin, leucomycin, picromycin), nucleosides (such as 5-azacytidine,puromycin, septacidin and amicetin; phenazines represented by myxin,lomofungin, iodin), oligosaccharides (including curamycin andeveminomycin; sulfonamides represented by sulfathiazole, sulfadiazine,sulfanilimide, sulfapyrazine) polyenes (including amphotericins,candicidin and nystatin, polyethers tetracyclines (includingdoxycyclines, minocyclines, methacylcines, chlortetracyclines,oxytetracylcines, demeclocylcines), nitrofurans (includingnitrofurazone, furazolidone, nitrofurantoin, furium, nitrovin andnifuroxime), and quinolone carboxylic acids (including nalidixic acid,piromidic acid, pipemidic acid and oxolinic acid); antiviral agentsincluding interferons α, β and γ, amantadine, rimantadine, arildone,ribaviran, acyclovir, abacavir, vidarabine (ARA-A)9-1,3-dihydroxy-2-propoxy methylguanine (DHPG), ganciclovir, enviroxime,foscarnet, ampligen, podophyllotoxin, 2,3-dideoxytidine (ddC),iododeoxyuridine (IDU), trifluorothymidine (TFT), dideoxyinosine (ddi),d4T, 3TC, zidovudine, efavirenz, protease inhibitors such as indinavir,saquinavir, ritonavir, nelfinavir, amprenavir, and specific antiviralantibodies; anti-cancer drugs, including cell cycle-specific agents(including structural analogs or antimetabolites of metholtrexate,mercaptopuorine, fluorouracil, cytarabine, thioguanine, azacitidine),bleomycin peptide antibiotics, such as podophyllin alkaloids includingetoposide (VP-16) and teniposide (VM-26), various plant alkaloids suchas vincristine, vinblastine, and paclitaxel, anti-neoplastic cellcycle-nonspecific agents such as various alkylating compounds such asbusulfan, cyclophosphamide, mechlorethamine, melphalan, altaretamine,ifosfamide, cisplatin, dacarbazine, procarbazine, lomustine, carmustine,lomustine, semustine, chlorambucil, thiotepa and carboplatin; varioushormones, hormone agonists and biologic response modifying agents,including flutamide, prednisone, ethinyl estradiol, diethylstilbestrol,hydroxyprogesterone caproate, medroxyprogesterone, megestrolacetate,testosterone, fluoxymesterone and thyroid hormones such as di-,tri- andtetraiodothyroidine, the aromatase inhibitor, amino glutethimide, thepeptide hormone inhibitor octreotide and gonadotropin-releasing hormoneagonists such as goserilin acetate and leuprolide, biologic responsemodifiers such as various cytokines, interferon alpha-2a, interferonalpha-2b, interferon-gamma, interferon-beta, interleukin-1,interleukin-2, interleukin-4, interleukin-10, monoclonal antibodies(anti-HER-2/neu humanized antibody), tumor necrosis factor,granulocyte-macrophage colony-stimulating factor,macrophage-colony-stimulating factor, various prostaglandins,phenylacetates, retinoic acids, leukotrines, thromboxanes and otherfatty acid derivatives; and radiation therapy.
 13. The method of claim1, wherein the mitochondrial uncoupling agent is an analog of 2,4dinitrophenol.
 14. The method of claim 1, wherein the mitochondrialuncoupling agent is a derivative of 2,4 dinitrophenol.
 15. A method forinducing intracellular free radicals comprising the step ofadministering an amount of a mitochondrial uncoupling agent sufficientto induce intracellular free radicals.
 16. The method of claim 15,wherein the mitochondrial uncoupling agent is 2,4 dinitrophenol.
 17. Themethod of claim 15, wherein the mitochondrial uncoupling agent isselected from the group consisting of: classic uncouplers, including 2,4dinitrophenol, clofazimine, albendazole, cambendazole, oxibendazole,triclabendazole (TCZ),6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole and theirsulfoxide and sulfone metabolites, thiobendazole, rafoxanide, bithionol,niclosamide, eutypine, various lichen acids (hydroxybenzoic acids) suchas (+)usnic acid, vulpinic acid and atranorin,2′,5-dichloro-3-t-butyl-4′-nitrosalicylanilide (S-13),3,4′,5-trichlorosalicylanilide (DCC), platanetin,2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799,AU-1421,3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradecin-1,7(8H)-dione(zearalenone), N,N′-bis-(4-trifluoromethylphenyl)-urea, resorcylic acidlactones and their derivatives,3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847),2,2,-bis(hexafluoroacetonyl)acetone, triphenyl boron, carbonylcyanide4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA), carbonylcyanide 3-chlorophenylhydrazone (CICCP), 1,3,6,8-tetranitrocarbazole,tetrachlorobenzotriazole, 4-iso-octyl-2,6-dinitrophenol(Octyl-DNP),4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxalbisguanylhydrazone), pentachlorophenol (PCP),5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI),N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid),4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol,2-azido-4-nitrophenol, 5-nitrobenzotriazole,5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole,methyl-o-phenylhydrazone, N-phenylanthranilic acid,N-(3-nitrophenyl)anthranilic acid, N-(2,3-dimethylphenyl)anthranilicacid, mefenamic acid, diflunisal, flufenamix acid,N-(3-chlorophenyl)anthranilic acid, carbonyl cyanide4-trifluoromethoxyphenylhydrazone (FCCP), SR-4233 (Tirapazamine),atovaquone, carbonyl cyanide4-(6′-methyl-2′-benzothiazyl)-phenylhydrazone (BT-CCP), ellipticine,olivacine, ellipticinium, isoellipticine and related isomers,methyl-O-phenylhydrazonocyanoaceticacid,methyl-O-(3-chlorophenylhydrazono)cyanoacetic acid,2-(3′-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic acid,2-(2′,4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile,relanium, melipramine, and other diverse chemical entities includingunsaturated fatty acids (up to C₁₄ optimum), sulflaramid and itsmetabolite perfluorooctane sulfonamide (DESFA), perfluorooctanoate,clofibrate, Wy-14, 643, ciprofibrate, and fluoroalcohols; ionophorousantibiotic uncouplers, including gramicidin, nigericin, tyrothricin,tyrocidin, valinomycin, alamethicins, harzianin HA V, saturnisporin SAIV, zervamicins, magainin, cecropins, melittin, hypelcins,suzukacillins, monensins, trichotoxins, antiamoebins, crystal violet,cyanine dyes, cadmium ion, trichosporin-B and their derivatives; andother heterogeneous uncoupling compounds, including desaspidin, ionizedcalcium (Ca⁺⁺), uncoupling proteins such as UCPI-1, UCP-2, UCP-3, PUMP(Plant Uncoupling Mitochondrial Protein), histones, polylysines,A206668-a protein, and compound K23187.
 18. The method of claim 15,wherein the mitochondrial uncoupling agent is a conjugate comprising 2,4dinitrophenol.
 19. The method of claim 15, wherein the mitochondrialuncoupling agent is a derivative of 2,4 dinitrophenol.
 20. The method ofclaim 15, wherein the mitochondrial uncoupling agent is a analog of 2,4dinitrophenol.
 21. The method of claim 15, further wherein the inducedintracellular free radicals are used in the diagnosis or treatment ofinfections, malignancies or other medical conditions.
 22. The method ofclaim 13, wherein the induced intracellular free radicals are used inthe diagnosis or treatment of infections, malignancies or other medicalconditions selected from the group consisting of cancer, and bacterial,parasitic, fungal, and viral infections or infestations.
 23. The methodof claim 21, wherein an animal is administered the mitochondrialuncoupling agent and a separate medication is administered, wherein thesecond medication increases the overall metabolic rate of the animal,the metabolic rate of a specific target tissue in the animal, or anincrease in free radical flux.
 24. The method of claim 23, wherein thesecond medication is selected from the group consisting of glucagon,arbutamine, dobutamine, vasopressin, glutamine, proline, octanoate,methylene blue (tetramethylthionine), ubiquinone, menadione,hematoprophyrin, polyunsaturated fatty acids including linoleic (doublebonds at carbons 9 and 12), alpha-linolenic (double bonds at carbons 9,12, and 15), gamma-linolenic (double bonds at carbons 6, 9, and 12),arachidonic (double bonds at carbons 5, 8, 11, and 14), eicosapentaenoic(double bonds at 5, 8, 11, 14, and 17), docosahexenoic (double bonds atcarbons 4, 7, 10, 13, 16, and 19), cis-parinaric (double bonds at 9, 11,13, and 15) and, monounsaturated fatty acids including oleic (doublebond at carbon 9), erucic (double bond at carbon 13), phenazinemethosulfate, 2,6-dichlorophenolindophenol, coenzyme Q1, CoQ2 and theiranalogs duroquinone and decylubiquinone.
 25. The method of claim 21wherein the intracellular free radicals are used in the diagnosis ortreatment of Lyme disease.
 26. The method of claim 21, wherein theinduced intracellular free radicals involve the induction of heat shockproteins.
 27. The method of claim 15, a second, therapeutic agent ortherapy is administered.
 28. The method of claim 27, wherein the second,therapeutic agent, or therapy, is selected from the group consisting of:anti-fungal agents, including Amphotericin B, Griseofulvin, Fluconazole(Diflucan), Intraconazole, 5 fluoro-cytosine (Flutocytosine, 5-FC),Ketatoconazole and Miconazole; anti-bacterial agents, including betalactam rings (penicillins), macrocyclic lactone rings (macrolides),polycyclic derivatives of napthacenecarboxamide (tetracyclines), aminosugars in glycosidic linkages (aminoglycosides), peptides (bacitracin,gramicedin, polymixins, etc.), nitrobenzene derivatives ofdichloroacedic acid, large ring compounds with conjugated double bondsystems (polyenes), various sulfa drugs including those derived fromsulfanilamide (sulfonamides, 5-nitro-2-furianyl compounds (nitrofurans),quinolone carboxylic acids (nalidixic acid), fluorinated quinilones(ciprofloxan, enoxacin, ofloxacin, etc.), nitroimidazoles(metroindazole), peptide antibiotics (such as bacitracin, bleomycin,cactinomycin, capreomycin, colistin, dactinomycin, gramacidin A,enduracitin, amphomycin, gramicidin J, mikamycins, polymyxins,stendomycin, actinomycin; aminoglycosides represented by streptomycin,neomycin, paromycin, gentamycin ribostamycin, tobramycin, amikacin;lividomycin beta lactams represented by benzylpenicillin, methicillin,oxacillin, hetacillin, piperacillin, amoxicillin and carbenacillin;lincosaminides represented by clindamycin, lincomycin, celesticetin,desalicetin; chloramphenicol; macrolides represented by erythromycins,lankamycin, leucomycin, picromycin), nucleosides (such as 5-azacytidine,puromycin, septacidin and amicetin; phenazines represented by myxin,lomofungin, iodin), oligosaccharides (including curamycin andeverninomycin; sulfonamides represented by sulfathiazole, sulfadiazine,sulfapyrazine) polyenes (including amphotericins, candicidin andnystatin, polyethers tetracyclines (including doxycyclines,minocyclines, methacylcines, chlortetracyclines, oxytetracylcines,demeclocylcines), nitrofurans (including nitrofurazone, furazolidone,nitrofurantoin, furium, nitrovin and nifuroxime), and quinolonecarboxylic acids (including nalidixic acid, piromidic acid, pipemidicacid and oxolinic acid); antiviral agents including interferons α, β andγ, amantadine, rimantadine, arildone, ribaviran, acyclovir, abacavir,vidarabine (ARA-A) 9-1,3-dihydroxy-2-propoxy methylguanine (DHPG),ganciclovir, enviroxime, foscarnet, ampligen, podophyllotoxin,2,3-dideoxytidine (ddC), iododeoxyuridine (IDU), trifluorothymidine(TFT), dideoxyinosine (ddi), d4T, 3TC, zidovudine, efavirenz, proteaseinhibitors such as indinavir, saquinavir, ritonavir, nelfinavir,amprenavir, and specific antiviral antibodies; anti-cancer drugs,including cell cycle-specific agents (including structural analogs orantimetabolites of metholtrexate, mercaptopuorine, fluorouracil,cytarabine, thioguanine, azacitidine), bleomycin peptide antibiotics,such as podophyllin alkaloids including etoposide (VP-16) and teniposide(VM-26), various plant alkaloids such as vincristine, vinblastine, andpaclitaxel, anti-neoplastic cell cycle-nonspecific agents such asvarious alkylating compounds such as busulfan, cyclophosphamide,mechlorethamine, melphalan, altaretamine, ifosfamide, cisplatin,dacarbazine, procarbazine, lomustine, carmustine, lomustine, semustine,chlorambucil, thiotepa and carboplatin; various hormones, hormoneagonists and biologic response modifying agents, including flutamide,prednisone, ethinyl estradiol, diethyltilbestrol, hydroxyprogesteronecaproate, medroxyprogesterone, megestrolacetate, testosterone,fluoxymesterone and thyroid hormones such as di-,tri- andtetraiodothyroidine, the aromatase inhibitor, amino glutethimide, thepeptide hormone inhibitor octreotide and gonadotropin-releasing hormoneagonists such as goserilin acetate and leuprolide, biologic responsemodifiers such as various cytokines, interferon alpha-2a, interferonalpha-2b, interferon-gamma, interferon-beta, interleukin-1,interleukin-2, interleukin-4, interleukin-10, monoclonal antibodies(anti-HER-2/neu humanized antibody), tumor necrosis factor,granulocyte-macrophage colony-stimulating factor,macrophage-colony-stimulating factor, various prostaglandins,phenylacetates, retinoic acids, leukotrines, thromboxanes and otherfatty acid derivatives; and radiation therapy.
 29. A method of treatingdisease in an animal by inducing intracellular hyperthermia comprisingthe step of administering an amount of a mitochondrial uncoupling agentsufficient to induce intracellular hyperthermia.
 30. The method of claim29, wherein the mitochondrial uncoupling agent is 2,4 dinitrophenol. 31.The method of claim 29, wherein the mitochondrial uncoupling agent is aconjugate comprising 2,4 dinitrophenol.
 32. The method of claim 29,wherein the disease is selected from the group consisting of cancer, andinfections or infestations of bacterial, parasitic, fungal, and viralpathogens.
 33. The method of claim 29, wherein the induced intracellularhyperthermia is used in the treatment of cancer.
 34. The method of claim29, wherein an animal is administered the mitochondrial uncoupling agentand a separate medication is administered, wherein the second medicationincreases the overall metabolic rate of the animal, the metabolic rateof a specific target tissue in the animal, or an increase in free radialflux.
 35. The method of claim 34, wherein the second medication isselected from the group consisting of glucagon, arbutamine, dobutamine,vasopressin, glutamine, proline, octanoate, methylene blue(tetramethylthionine), ubiquinone, menadione, hematoprophyrin,polyunsaturated fatty acids including linoleic (double bonds at carbons9 and 12), alpha-linolenic (double bonds at carbons 9, 12, and 15),gamma-linolenic (double bonds at carbons 6, 9, and 12), arachidonic(double bonds at carbons 5, 8, 11, and 14), eicosapentaenoic (doublebonds at 5, 8, 11, 14, and 17), docosahexenoic (double bonds at carbons4, 7, 10, 13, 16, and 19), cis-parinaric (double bonds at 9, 11, 13, and15) and, monounsaturated fatty acids including oleic (double bond atcarbon 9), erucic (double bond at carbon 13), phenazine methosulfate,2,6-dichlorophenolindophenol, coenzyme Q1, CoQ2 and their analogsduroquinone and decylubiquinone.
 36. The method of claim 29, wherein theinduced intracellular hyperthermia used involves the induction of heatshock proteins.
 37. A method for diagnosing disease in an animal bychemically inducing intracellular hyperthermia comprising the step ofadministering an amount of a mitochondrial uncoupling agent sufficientto induce intracellular hyperthermia.
 38. The method of claim 37,wherein the mitochondrial uncoupling agent is 2,4 dinitrophenol.
 39. Themethod of claim 37, wherein the mitochondrial uncoupling agent is aconjugate comprising 2,4 dinitrophenol.
 40. The method of claim 37,wherein the disease is selected from the group consisting of cancer, andinfections or infestations of bacterial, parasitic, fungal, and viralpathogens.
 41. The method of claim 37, wherein the induced intracellularhyperthermia is used in the diagnosis or treatment of cancer.
 42. Themethod of claim 37, wherein an animal is administered the mitochondrialuncoupling agent and a separate medication is administered, wherein thesecond medication increases the overall metabolic rate of the animal,the metabolic rate of a specific target tissue in the animal, or anincrease in free radical flux.
 43. The method of claim 42, wherein thesecond medication is selected from the group consisting of glucagon,arbutamine, dobutamine, vasopressin, glutamine, proline, octanoate,methylene blue (tetramethylthionine), ubiquinone, menadione,hematoprophyrin, polyunsaturated fatty acids including linoleic (doublebonds at carbons 9 and 12), alpha-linolenic (double bonds at carbons 9,12, and 15), gamma-linolenic (double bonds at carbons 6, 9, and 12),arachidonic (double bonds at carbons 5, 8, 11, and 14), eicosapentaenoic(double bonds at 5, 8, 11, 14, and 17), docosahexenoic (double bonds atcarbons 4, 7, 10, 13, 16, and 19), cis-parinaric (double bonds at 9, 11,13, and 15) and, monounsaturated fatty acids including oleic (doublebond at carbon 9), erucic (double bond at carbon 13), phenazinemethosulfate, 2,6-dichlorophenolindophenol, coenzyme Q1, CoQ2 and theiranalogs duroquinone and decylubiquinone
 44. The method of claim 37,wherein the induced intracellular hyperthermia involves the induction ofheat shock proteins.
 45. The method of claim 37, wherein themitochondrial uncoupling agent is selected from the group consisting of:classic uncouplers, including 2,4 dinitrophenol, clofazimine,albendazole, cambendazole, oxibendazole, triclabendazole (TCZ),6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole and theirsulfoxide and sulfone metabolites, thiobendazole, rafoxanide, bithionol,niclosamide, eutypine, various lichen acids (hydroxybenzoic acids) suchas (+)usnic acid, vulpinic acid and atranorin,2′,5-dichloro-3-t-butyl-4′-nitrosalicylanilide (S-13),3,4′,5-trichlorosalicylanilide (DCC), platanetin,2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799,AU-1421,3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradecin-1,7(8H)-dione(zearalenone), N,N¹-bis-(4-trifluoromethylphenyl)-urea, resorcylic acidlactones and their derivatives,3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847),2,2,-bis(hexafluoroacetonyl)acetone, triphenyl boron, carbonylcyanide4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA), carbonylcyanide 3-chlorophenylhydrazone (ClCCP), 1,3,6,8-tetranitrocarbazole,tetrachlorobenzotriazole, 4-iso-octyl-2,6-dinitrophenol(Octyl-DNP),4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxalbisguanylhydrazone), pentachlorophenol (PCP),5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI),N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid),4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol,2-azido-4-nitrophenol, 5-nitrobenzotriazole,5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole,methyl-o-phenylhydrazone, N-phenylanthranilic acid,N-(3-nitrophenyl)anthranilic acid, N-(2,3-dimethylphenyl)anthranilicacid, mefenamic acid, diflunisal, flufenamix acid,N-(3-chlorophenyl)anthranilic acid, carbonyl cyanide4-trifluoromethoxyphenylhydrazone (FCCP), SR-4233 (Tirapazamine),atovaquone, carbonyl cyanide4-(6′-methyl-2′-benzothiazyl)-phenylhydrazone (BT-CCP), ellipticine,olivacine, ellipticinium, isoellipticine and related isomers,methyl-O-phenylhydrazonocyanoaceticacid,methyl-O-(3-chlorophenylhydrazono)cyanoacetic acid,2-(3′-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic acid,2-(2′,4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile,relanium, melipramine, and other diverse chemical entities includingunsaturated fatty acids (up to C₁₄ optimum), sulflaramid and itsmetabolite perfluorooctane sulfonamide (DESFA), perfluorooctanoate,clofibrate, Wy-14, 643, ciprofibrate, and fluoroalcohols; ionophorousantibiotic uncouplers, including gramicidin, nigericin, tyrothricin,tyrocidin, valinomycin, alamethicins, harzianin HA V, saturnisporin SAIV, zervamicins, magainin, cecropins, melittin, hypelcins,suzukacillins, monensins, trichotoxins, antiamoebins, crystal violet,cyanine dyes, cadmium ion, trichosporin-B and their derivatives; andother heterogeneous uncoupling compounds, including desaspidin, ionizedcalcium (Ca⁺⁺), uncoupling proteins such as UCPI-1, UCP-2, UCP-3, PUMP(Plant Uncoupling Mitochondrial Protein), histones, polylysines,A206668-a protein, and compound K23187.
 46. The method of claim 37,wherein a second therapeutic agent or therapy is administered.
 47. Themethod of claim 46, wherein the second therapeutic agent or therapy isselected from the group consisting of: anti-fungal agents, includingAmphotericin B, Griseofulvin, Fluconazole (Diflucan), Intraconazole, 5fluoro-cytosine (Flutocytosine, 5-FC), Ketatoconazole and Miconazole;anti-bacterial agents, including beta lactam rings (penicillins),macrocyclic lactone rings (macrolides), polycyclic derivatives ofnapthacenecarboxamide (tetracyclines), amino sugars in glycosidiclinkages (aminoglycosides), peptides (bacitracin, gramicedin,polymixins, etc.), nitrobenzene derivatives of dichloroacedic acid,large ring compounds with conjugated double bond systems (polyenes),various sulfa drugs including those derived from sulfanilamide(sulfonamides, 5-nitro-2-furianyl compounds (nitrofurans), quinolonecarboxylic acids (nalidixic acid), fluorinated quinilones (ciprofloxan,enoxacin, ofloxacin, etc.), nitroimidazoles (metroindazole), peptideantibiotics (such as bacitracin, bleomycin, cactinomycin, capreomycin,colistin, dactinomycin, gramacidin A, enduracitin, amphomycin,gramicidin J, mitramycins, polymyxins, stendomycin, actinomycin;aminoglycosides represented by streptomycin, neomycin, paromycin,gentamycin ribostamycin, tobramycin, amikacin; lividomycin beta lactamsrepresented by benzylpenicillin, methicillin, oxacillin, hetacillin,piperacillin, amoxicillin and carbenacillin; lincosaminides representedby clindamycin, lincomycin, celesticetin, desalicetin; chloramphenicol;macrolides represented by erythromycins, lankamycin, leucomycin,picromycin), nucleosides (such as 5-azacytidine, puromycin, septacidinand amicetin; phenazines represented by myxin, lomofungin, iodin),oligosaccharides (including curamycin and everninomycin; sulfonamidesrepresented by sulfathiazole, sulfadiazine, sulfanilimide,sulfapyrazine) polyenes (including amphotericins, candicidin andnystatin, polyethers tetracyclines (including doxycyclines,minocyclines, methacylcines, chlortetracyclines, oxytetracylcines,demeclocylcines), nitrofurans (including nitrofurazone, furazolidone,nitrofurantoin, furium, nitrovin and nifuroxime), and quinolonecarboxylic acids (including nalidixic acid, piromidic acid, pipemidicacid and oxolinic acid); antiviral agents including interferons α, β andγ, amantadine, rimantadine, arildone, ribaviran, acyclovir, abacavir,vidarabine (ARA-A) 9-1,3-dihydroxy-2-propoxy methylguanine (DHPG),ganciclovir, enviroxime, foscarnet, ampligen, podophyllotoxin,2,3-dideoxytidine (ddC), iododeoxyuridine (IDU), trifluorothymidine(TFT), dideoxyinosine (ddi), d4T, 3TC, zidovudine, efavirenz, proteaseinhibitors such as indinavir, saquinavir, ritonavir, nelfinavir,amprenavir, and specific antiviral antibodies; anti-cancer drugs,including cell cycle-specific agents (including structural analogs orantimetabolites of metholtrexate, mercaptopuorine, fluorouracil,cytarabine, thioguanine, azacitidine), bleomycin peptide antibiotics,such as podophyllin alkaloids including etoposide (VP-16) and teniposide(VM-26), various plant alkaloids such as vincristine, vinblastine, andpaclitaxel, anti-neoplastic cell cycle-nonspecific agents such asvarious alkylating compounds such as busulfan, cyclophosphamide,mechlorethamine, melphalan, altaretamine, ifosfamide, cisplatin,dacarbazine, procarbazine, lomustine, carmustine, lomustine, semustine,chlorambucil, thiotepa and carboplatin; various hormones, hormoneagonists and biologic response modifying agents, including flutamide,prednisone, ethinyl estradiol, diethylstilbestrol, hydroxyprogesteronecaproate, medroxyprogesterone, megestrolacetate, testosterone,fluoxymesterone and thyroid hormones such as di-,tri- andtetraiodothyroidine, the aromatase inhibitor, amino glutethimide, thepeptide hormone inhibitor octreotide and gonadotropin-releasing hormoneagonists such as goserilin acetate and leuprolide, biologic responsemodifiers such as various cytokines, interferon alpha-2a, interferonalpha-2b, interferon-gamma, interferon-beta, interleukin-1,interleukin-2, interleukin-4, interleukin-10, monoclonal antibodies(anti-HER-2/neu humanized antibody), tumor necrosis factor,granulocyte-macrophage colony-stimulating factor,macrophage-colony-Stimulating factor, various prostaglandins,phenylacetates, retinoic acids, leukotrines, thromboxanes and otherfatty acid derivatives; and radiation therapy.
 48. A method of inducingheat shock proteins in an animal comprising the step of administeringamount of a mitochondrial uncoupling agent sufficient to induce heatshock proteins.
 49. The method of claim 48, wherein the mitochondrialuncoupling agent is 2,4 dinitrophenol.
 50. The method of claim 48,wherein the mitochondrial uncoupling agent is selected from the groupconsisting of: classic uncouplers, including 2,4 dinitrophenol,clofazimine, albendazole, cambendazole, oxibendazole, triclabendazole(TCZ), 6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole andtheir sulfoxide and sulfone metabolites, thiobendazole, rafoxanide,bithionol, niclosamide, eutypine, various lichen acids (hydroxybenzoicacids) such as (+)usnic acid, vulpinic acid and atranorin,2′,5-dichloro-3-t-butyl-4′-nitrosalicylanilide (S-13),3,4′,5-trichlorosalicylanilide (DCC), platanetin,2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799,AU-1421,3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradecin-1,7(8H)-dione(zearalenone), N,N¹-bis-(4-trifluoromethylphenyl)-urea, resorcylic acidlactones and their derivatives,3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847),2,2,-bis(hexafluoroacetonyl)acetone, triphenyl boron, carbonylcyanide4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA), carbonylcyanide 3-chlorophenylhydrazone (ClCCP), 1,3,6,8-tetranitrocarbazole,tetrachlorobenzotriazole, 4-iso-octyl-2,6-dinitrophenol(Octyl-DNP),4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxalbisguanylhydrazone), pentachlorophenol (PCP),5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI),N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid),4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol,2-azido-4-nitrophenol, 5-nitrobenzotriazole,5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole,methyl-o-phenylhydrazone, N-phenylanthranilic acid,N-(3-nitrophenyl)anthranilic acid, N-(2,3-dimethylphenyl)anthranilicacid, mefenamic acid, diflunisal, flufenamix acid,N-(3-chlorophenyl)anthranilic acid, carbonyl cyanide4-trifluoromethoxyphenylhydrazone (FCCP), SR-4233 (Tirapazamine),atovaquone, carbonyl cyanide4-(6′-methyl-2′-benzothiazyl)-phenylhydrazone (BT-CCP), ellipticine,olivacine, ellipticinium, isoellipticine and related isomers,methyl-O-phenylhydrazonocyanoaceticacid,methyl-O-(3-chlorophenylhydrazono)cyanoacetic acid,2-(3′-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic acid,2-(2′,4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile,relanium, melipramine, and other diverse chemical entities includingunsaturated fatty acids (up to C₁₄ optimum), sulflaramid and itsmetabolite perfluorooctane sulfonamide (DESFA), perfluorooctanoate,clofibrate, Wy-14, 643, ciprofibrate, and fluoroalcohols; ionophorousantibiotic uncouplers, including gramicidin, nigericin, tyrothricin,tyrocidin, valinomycin, alamethicins, harzianin HA V, saturnisporin SAIV, zervamicins, magainin, cecropins, melittin, hypelcins,suzukacillins, monensins, trichotoxins, antiamoebins, crystal violet,cyanine dyes, cadmium ion, trichosporin-B and their derivatives; andother heterogeneous uncoupling compounds, including desaspidin, ionizedcalcium (Ca⁺⁺), uncoupling proteins such as UCPI-1, UCP-2, UCP-3, PUMP(Plant Uncoupling Mitochondrial Protein), histones, polylysines,A206668-a protein, and compound K23187.
 51. The method of claim 48,wherein the mitochondrial uncoupling agent is a conjugate comprising 2,4dinitrophenol.
 52. The method of claim 48, further wherein the inducedheat shock proteins condition the animal for a specific condition. 53.The method of claim 52, wherein the specific condition is surgery. 54.The methods of claims 1, 15, 29, 48 and 51, wherein the uncoupling agentis produced using combinatorial technology.